Regulation of stanniocalcin-1 secretion by BeWo cells and first trimester human placental tissue from normal pregnancies and those at increased risk of developing preeclampsia.

Stanniocalcin-1 (STC-1) is a multi-functional glycosylated peptide present in the plasma of healthy women postpartum and increased further in pregnancies complicated by preeclampsia. Although the STC-1 gene is expressed by the placenta what regulates its secretion and from which cells at the feto-maternal interface is unknown. Here, we demonstrate for the first time that the syncytiotrophoblast and cytotrophoblast are a major site of STC-1 protein expression in first trimester placental tissue. Further, in response to low oxygen, first trimester chorionic villous tissue from pregnancies at increased risk of developing preeclampsia secreted significantly more STC-1 than normal tissue under the same conditions. Using the human trophoblast cell line BeWo we have shown that low oxygen increased the secretion of STC-1 but it required co-stimulation with the Adenosine-3', 5'-cyclic monophosphate (cAMP) analogue, 8-Bromo adenosine-3', 5'-cyclic monophosphate cAMP (8 Br-cAMP) to reach significance. Inhibition of Hypoxia inducible factor 2α (HIF-2α) and the Phosphatidylinositol-3 kinase (PI3 -Kinase)/AKT/Serum and glucocorticoid-induced kinase-1(SGK-1) pathway resulted in significant inhibition of STC-1 secretion. As both low oxygen and cAMP are known to play a central role in placental function, their regulation of STC-1 points to a potentially important role in the maintenance of a normal healthy pregnancy and we would hypothesize that it may act to protect against prolonged placental hypoxia seen in preeclampsia.

First trimester placental endothelial cells from pregnancies with abnormal uterine artery Doppler are more sensitive to apoptotic stimuli.

Failure of the placental capillary network to develop normally is associated with early onset fetal growth restriction (FGR) and pre-eclampsia (PE). Although the symptoms are observed at term, the problem begins in the first trimester. However, investigations at this clinically relevant time are hindered by difficulties in identifying earlystage pregnancies that are at risk of developing FGR/PE. Using uterine artery Doppler ultrasound in the first trimester as a proxy measure of poor placentation, we have identified pregnancies at increased risk of developing early onset FGR/PE. Placental endothelial cells (PEC) isolated from pregnancies at increased risk of developing FGR/PE grew more slowly and their basal rate of apoptosis was significantly higher than that seen in the normal group. The pro-apoptotic stimulus, TNFα, induced apoptosis in cells from both groups but this was significantly greater in the high risk group. TNF receptor expression was unaffected. Inhibition of nitric oxide (NO) production significantly increased the sensitivity of cells from the normal pregnancies to TNFα but not in the high risk group establishing a functional role for NO in this system. In conclusion, first trimester PEC from pregnancies at increased risk of developing early onset FGR/PE were inherently more sensitive to apoptotic stimuli and this was functionally linked to the synthesis of NO. This may contribute to the poor placental vascular development seen in on going pregnancies.

Disturbed Placental Imprinting in Preeclampsia Leads to Altered Expression of DLX5, a Human-Specific Early Trophoblast Marker.

BACKGROUND: Preeclampsia is a complex and common human-specific pregnancy syndrome associated with placental pathology. The human specificity provides both intellectual and methodological challenges, lacking a robust model system. Given the role of imprinted genes in human placentation and the vulnerability of imprinted genes to loss of imprinting changes, there has been extensive speculation, but no robust evidence, that imprinted genes are involved in preeclampsia. Our study aims to investigate whether disturbed imprinting contributes to preeclampsia. METHODS: We first aimed to confirm that preeclampsia is a disease of the placenta by generating and analyzing genome-wide molecular data on well-characterized patient material. We performed high-throughput transcriptome analyses of multiple placenta samples from healthy controls and patients with preeclampsia. Next, we identified differentially expressed genes in preeclamptic placentas and intersected them with the list of human imprinted genes. We used bioinformatics/statistical analyses to confirm association between imprinting and preeclampsia and to predict biological processes affected in preeclampsia. Validation included epigenetic and cellular assays. In terms of human specificity, we established an in vitro invasion-differentiation trophoblast model. Our comparative phylogenetic analysis involved single-cell transcriptome data of human, macaque, and mouse preimplantation embryogenesis. RESULTS: We found disturbed placental imprinting in preeclampsia and revealed potential candidates, including GATA3 and DLX5, with poorly explored imprinted status and no prior association with preeclampsia. As a result of loss of imprinting, DLX5 was upregulated in 69% of preeclamptic placentas. Levels of DLX5 correlated with classic preeclampsia markers. DLX5 is expressed in human but not in murine trophoblast. The DLX5high phenotype resulted in reduced proliferation, increased metabolism, and endoplasmic reticulum stress-response activation in trophoblasts in vitro. The transcriptional profile of such cells mimics the transcriptome of preeclamptic placentas. Pan-mammalian comparative analysis identified DLX5 as part of the human-specific regulatory network of trophoblast differentiation. CONCLUSIONS: Our analysis provides evidence of a true association among disturbed imprinting, gene expression, and preeclampsia. As a result of disturbed imprinting, the upregulated DLX5 affects trophoblast proliferation. Our in vitro model might fill a vital niche in preeclampsia research. Human-specific regulatory circuitry of DLX5 might help explain certain aspects of preeclampsia.

Increased apoptosis, altered oxygen signaling, and antioxidant defenses in first-trimester pregnancies with high-resistance uterine artery blood flow.

The mechanisms of deficient placentation in the first trimester remain poorly understood, although apoptosis, hypoxia, and oxidative stress have been implicated. High uterine artery Doppler resistance indexes (RIs) are predictive of placental complications of pregnancy, such as preeclampsia, fetal growth restriction, and stillbirth. We provide evidence that even in the first trimester, pregnancies with high uterine artery Doppler RI demonstrate alterations in placental gene and protein expression. Apoptosis was significantly higher in high RI placental tissue, as determined by Western blot analysis of cleaved poly (ADP-ribose) polymerase and caspase 3. Protein expression of the trophoblast survival factor insulin-like growth factor-2 was significantly lower. Both high and normal RI placentas showed evidence of hypoxia and oxidative stress with expression of hypoxia-inducible factors 1α and 2α, heat shock protein 70, presence of nitrotyrosine residues, and lipid peroxidation. We observed no exaggerated placental hypoxia or oxidative stress associated with high RI pregnancies. High RI placental tissue demonstrated an altered balance of antioxidant enzyme activity. Hypoxia and oxidative stress appear to be a physiological state in early pregnancy; our data did not support the hypothesis that they are associated with deficient placentation in the first trimester. Higher levels of apoptosis, reduced insulin-like growth factor-2 expression, and altered antioxidant defenses may contribute to abnormal placentation and the later development of pregnancy complications, such as preeclampsia, fetal growth restriction, and stillbirth.

Homeobox gene TGIF-1 is increased in placental endothelial cells of human fetal growth restriction.

Aberrant placental angiogenesis is associated with fetal growth restriction (FGR). In mice, targeted disruption of the homeobox gene, transforming growth ß-induced factor (Tgif-1), which is also a transcription factor, causes defective placental vascularisation. Nevertheless, the role of TGIF-1 in human placental angiogenesis is unclear. We have previously reported increased TGIF-1 expression in human FGR placentae and demonstrated localisation of TGIF-1 protein in placental endothelial cells (ECs). However, its functional role remains to be investigated. In this study, we aimed to specifically compare TGIF-1 mRNA expression in placental ECs isolated from human FGR-affected pregnancies with gestation-matched control pregnancies in two independent cohorts from Australia and Canada and to identify the functional role of TGIF-1 in placental angiogenesis using the human umbilical vein endothelial cell-derived cell line, SGHEC-7, and primary human umbilical vein ECs. Real-time PCR revealed that TGIF-1 mRNA expression was significantly increased in ECs isolated from FGR-affected placentae compared with that of controls. The functional roles of TGIF-1 were determined in ECs after TGIF-1 siRNA transfection. TGIF-1 inactivation in ECs significantly reduced TGIF-1 at both the mRNA and protein levels, as well as the proliferative and invasive potential, but significantly increased the angiogenic potential. Using angiogenesis PCR screening arrays, we identified ITGAV, NRP-1, ANPGT-1 and ANPGT-2 as novel downstream targets of TGIF-1, after TGIF-1 inactivation in ECs. Collectively, these results show that TGIF-1 regulates EC function and the expression of angiogenic molecules; and when abnormally expressed, may contribute to the aberrant placental angiogenesis observed in FGR.

Oxygen modulates human decidual natural killer cell surface receptor expression and interactions with trophoblasts.

Decidual natural killer (dNK) cells have been shown to both promote and inhibit trophoblast behavior important for decidual remodeling in pregnancy and have a distinct phenotype compared to peripheral blood NK cells. We investigated whether different levels of oxygen tension, mimicking the physiological conditions of the decidua in early pregnancy, altered cell surface receptor expression and activity of dNK cells and their interactions with trophoblast. dNK cells were isolated from terminated first-trimester pregnancies and cultured in oxygen tensions of 3%, 10%, and 21% for 24 h. Cell surface receptor expression was examined by flow cytometry, and the effects of secreted factors in conditioned medium (CM) on the trophoblast cell line SGHPL-4 were assessed in vitro. SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 10% were significantly more invasive (P < 0.05) and formed endothelial-like networks to a greater extent (P < 0.05) than SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 3% or 21%. After 24 h, a lower percentage of dNK cells expressed CD56 at 21% oxygen (P < 0.05), and an increased percentage of dNK cells expressed NKG2D at 10% oxygen (P < 0.05) compared to other oxygen tensions, with large patient variation. This study demonstrates dNK cell phenotype and secreted factors are modulated by oxygen tension, which induces changes in trophoblast invasion and endovascular-like differentiation. Alterations in dNK cell surface receptor expression and secreted factors at different oxygen tensions may represent regulation of function within the decidua during the first trimester of pregnancy.

Decidual natural killer cell interactions with trophoblasts are impaired in pregnancies at increased risk of preeclampsia.

Transformation of the uterine spiral arteries (SAs) during pregnancy is critical to support the developing fetus, and is impaired in some pregnancy disorders, including preeclampsia. Decidual natural killer (dNK) cells play a role in SA remodeling, although their interactions with fetal trophoblast remain unclear. A uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure of the extent of SA remodeling; we have used this technique to characterize dNK cells from pregnancies with normal (normal RI) and impaired (high RI) SA remodeling, which display least and highest risk of developing preeclampsia, respectively. We examined the impact of dNK cell secreted factors on trophoblast motility, chemoattraction, and signaling pathways to determine the contribution of dNK cells to SA transformation. We demonstrated that the chemoattraction of the trophoblast by dNK cells is impaired in pregnancies with high RI, as is the ability to induce trophoblast outgrowth from placental villous explants. These processes are dependent on activation of the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase-Akt signaling pathways, which were altered in trophoblasts incubated with secreted factors from dNK cells from high RI pregnancies. Therefore, by characterizing pregnancies using uterine artery Doppler RI before dNK cell isolation, we have identified that impaired dNK-trophoblast interactions may lead to poor placentation. These findings have implications for pregnancy pathological conditions, such as preeclampsia.

Trophoblast-induced changes in C-x-C motif chemokine 10 expression contribute to vascular smooth muscle cell dedifferentiation during spiral artery remodeling.

OBJECTIVE: During pregnancy, fetal trophoblast disrupt endothelial cell and vascular smooth muscle cell (VSMC) interactions in spiral arteries of the maternal decidua to enable increased nutritional and oxygen delivery to the fetus. Little is known regarding this transformation because of difficulties of studying human pregnancy in vivo. This study investigated how trophoblast-secreted factors affect the interactions of vascular cells and the differentiation status of VSMC during spiral arteries remodeling using 3-dimensional vascular spheroid coculture. METHODS AND RESULTS: Endothelial cell and VSMC were cocultured in hanging droplets to form spheroids representing an inverted vessel lumen. Control or conditioned media from an extravillous trophoblast (EVT) cell line was incubated with vascular spheroids for 24 hours. Spheroid RNA was then analyzed by Illumina Sentrix BeadChip array. Spheroids incubated with EVT conditioned medium showed significant up/downregulation of 101 genes (>1.5-fold; P<0.05), including an upregulation of C-X-C motif chemokine 10 (IP-10). C-X-C motif chemokine 10 expression was confirmed by qualitative real-time PCR and Western blot analysis of spheroids, and immunohistochemistry of first trimester decidua and ex vivo dissected nonplacental bed spiral arteries. EVT conditioned medium reduced VSMC expression of differentiation markers, and both EVT conditioned medium and C-X-C motif chemokine 10 increased motility of VSMC indicating dedifferentiation of VSMC. CONCLUSIONS: EVT-induced C-X-C motif chemokine 10 expression may contribute to spiral arteries remodeling during pregnancy by altering the motility and differentiation status of the VSMC in the vessel.

Role of the eIF4E binding protein 4E-BP1 in regulation of the sensitivity of human pancreatic cancer cells to TRAIL and celastrol-induced apoptosis.

BACKGROUND INFORMATION: Tumour cells can be induced to undergo apoptosis after treatment with the tumour necrosis factor α-related death-inducing ligand (TRAIL). Although human pancreatic cancer cells show varying degrees of response they can be sensitised to the pro-apoptotic effects of TRAIL in the presence of celastrol, a natural compound extracted from the plant Tripterygium wilfordii Hook F. One important aspect of the cellular response to TRAIL is the control of protein synthesis, a key regulator of which is the eukaryotic initiation factor 4E-binding protein, 4E-BP1. RESULTS: We examined the effects of celastrol and TRAIL in several pancreatic cancer cell lines. In cells that are normally resistant to TRAIL, synergistic effects of TRAIL plus celastrol on commitment to apoptosis and inhibition of protein synthesis were observed. These were associated with a strong up-regulation and dephosphorylation of 4E-BP1. The enhancement of 4E-BP1 expression, which correlated with a threefold increase in the level of the 4E-BP1 transcript, was blocked by inhibitors of reactive oxygen species and the JNK protein kinase. When the expression of 4E-BP1 was reduced by an inducible micro-RNA, TRAIL-mediated apoptosis was inhibited. CONCLUSION: These results suggest that 4E-BP1 plays a critical role in the mechanism by which TRAIL and celastrol together cause apoptotic cell death in human pancreatic tumour cells.

Impaired decidual natural killer cell regulation of vascular remodelling in early human pregnancies with high uterine artery resistance.

During human pregnancy, natural killer (NK) cells accumulate in the maternal decidua, but their specific roles remain to be determined. Decidual NK (dNK) cells are present during trophoblast invasion and uterine spiral artery remodelling. These events are crucial for successful placentation and the provision of an adequate blood supply to the developing fetus. Remodelling of spiral arteries is impaired in the dangerous pregnancy complication pre-eclampsia. We studied dNK cells isolated from pregnancies at 9-14 weeks' gestation, screened by uterine artery Doppler ultrasound to determine resistance indices which relate to the extent of spiral artery remodelling. dNK cells were able to promote the invasive behaviour of fetal trophoblast cells, partly through HGF. Cells isolated from pregnancies with higher resistance indices were less able to do this and secreted fewer pro-invasive factors. dNK cells from pregnancies with normal resistance indices could induce apoptotic changes in vascular smooth muscle and endothelial cells in vitro, events of importance in vessel remodelling, partly through Fas signalling. dNK cells isolated from high resistance index pregnancies failed to induce vascular apoptosis and secreted fewer pro-apoptotic factors. We have modelled the cellular interactions at the maternal-fetal interface and provide the first demonstration of a functional role for dNK cells in influencing vascular cells. A potential mechanism contributing to impaired vessel remodelling in pregnancies with a higher uterine artery resistance is presented. These findings may be informative in determining the cellular interactions contributing to the pathology of pregnancy disorders where remodelling is impaired, such as pre-eclampsia.

Trophoblast- and vascular smooth muscle cell-derived MMP-12 mediates elastolysis during uterine spiral artery remodeling.

During the first trimester of pregnancy, the uterine spiral arteries are remodeled, creating heavily dilated conduits that lack maternal vasomotor control but allow the placenta to meet an increasing requirement for nutrients and oxygen. To effect permanent vasodilatation, the internal elastic lamina and medial elastin fibers must be degraded. In this study, we sought to identify the elastolytic proteases involved in this process. Primary first-trimester cytotrophoblasts (CTBs) derived from the placenta exhibited intracellular and membrane-associated elastase activity; membrane-associated activity was primarily attributable to matrix metalloproteinases (MMP). Indeed, Affymetrix microarray analysis and immunocytochemistry implicated MMP-12 (macrophage metalloelastase) as a key mediator of elastolysis. Cultured human aortic smooth muscle cells (HASMCs) exhibited constitutive membrane-associated elastase activity and inducible intracellular elastase activity; these cells also expressed MMP-12 protein. Moreover, a specific inhibitor of MMP-12 significantly reduced CTB- and HASMC-mediated elastolysis in vitro, to 31.7 ± 10.9% and 23.3 ± 8.7% of control levels, respectively. MMP-12 is expressed by both interstitial and endovascular trophoblasts in the first-trimester placental bed and by vascular SMCs (VSMCs) in remodeling spiral arteries. Perfusion of isolated spiral artery segments with CTB-conditioned medium stimulated MMP-12 expression in medial VSMCs. Our data support a model in which trophoblasts and VSMCs use MMP-12 cooperatively to degrade elastin during vascular remodeling in pregnancy, with the localized release of elastin peptides and CTB-derived factors amplifying elastin catabolism.

Pre-eclampsia: fitting together the placental, immune and cardiovascular pieces.

The success of pregnancy is a result of countless ongoing interactions between the placenta and the maternal immune and cardiovascular systems. Pre-eclampsia is a serious pregnancy complication that arises from multiple potential aberrations in these systems. The pathophysiology of pre-eclampsia is established in the first trimester of pregnancy, when a range of deficiencies in placentation affect the key process of spiral artery remodelling. As pregnancy progresses to the third trimester, inadequate spiral artery remodelling along with multiple haemodynamic, placental and maternal factors converge to activate the maternal immune and cardiovascular systems, events which may in part result from increased shedding of placental debris. As we understand more about the pathophysiology of pre-eclampsia, it is becoming clear that the development of early- and late-onset pre-eclampsia, as well as intrauterine growth restriction (IUGR), does not necessarily arise from the same underlying pathology.

Stanniocalcin-1 in the female reproductive system and pregnancy.

BACKGROUND: Stanniocalcin-1 (STC-1) is a widely expressed glycoprotein hormone involved in a diverse spectrum of physiological and pathophysiological processes including angiogenesis, mineral homeostasis, cell proliferation, inflammation and apoptosis. Over the last 20 years, numerous studies have reported STC-1 expression within female reproductive tissues including the uterus, ovaries and placenta and implicated STC-1 in processes such as ovarian follicular development, blastocyst implantation, vascular remodelling in early pregnancy and placental development. Notably, dysregulation of STC-1 within reproductive tissues has been linked to the onset of severe reproductive disorders including endometriosis, polycystic ovary syndrome, poor trophoblast invasion and placental perfusion in early pregnancy. Furthermore, significant changes in tissue expression and in maternal systemic concentration take place throughout pregnancy and further substantiate the vital role of this protein in reproductive health and disease. OBJECTIVE AND RATIONALE: Our aim is to provide a comprehensive overview of the existing literature, to summarise the expression profile and roles of STC-1 within the female reproductive system and its associated pathologies. We highlight the gaps in the current knowledge and suggest potential avenues for future research. SEARCH METHODS: Relevant studies were identified through searching the PubMed database using the following search terms: 'stanniocalcin-1', 'placenta', 'ovary', 'endometrium', 'pregnancy', 'reproduction', 'early gestation'. Only English language papers published between 1995 and 2020 were included. OUTCOMES: This review provides compelling evidence of the vital function that STC-1 plays within the female reproductive system. The literature presented summarise the wide expression profile of STC-1 within female reproductive organs, as well as highlighting the putative roles of STC-1 in various functions in the reproductive system. Moreover, the observed link between altered STC-1 expression and the onset of various reproductive pathologies is presented, including those in pregnancy whose aetiology occurs in the first trimester. This summary emphasises the requirement for further studies on the mechanisms underlying the regulation of STC-1 expression and function. WIDER IMPLICATIONS: STC-1 is a pleiotropic hormone involved in the regulation of a number of important biological functions needed to maintain female reproductive health. There is also growing evidence that dysregulation of STC-1 is implicated in common reproductive and obstetric disorders. Greater understanding of the physiology and biochemistry of STC-1 within the field may therefore identify possible targets for therapeutic intervention and/or diagnosis.

Maternal Angiotensin Increases Placental Leptin in Early Gestation via an Alternative Renin-Angiotensin System Pathway: Suggesting a Link to Preeclampsia.

[Figure: see text].

Remodelling at the maternal-fetal interface: relevance to human pregnancy disorders.

In human pregnancy, successful placentation and remodelling of the uterine vasculature require the integration of a number of stages, which are crucial for a healthy pregnancy. As the demands of the developing fetus for nutrients and oxygen increase, the capacity of the maternal blood vessels to supply this must be altered radically, with deficiencies in this process implicated in a number of dangerous pregnancy complications. The complex signalling networks that regulate these tightly co-ordinated events are becoming clearer as more studies of early pregnancy are performed. It is the aim of this review to draw together our knowledge of events that occur to facilitate a successful pregnancy ranging from the preparation for implantation, through the invasion and differentiation of the trophoblast and the regulation of these processes by other cells within the decidual environment, to the active role that the trophoblast and maternal immune cells play in facilitating the remodelling of the uterine spiral arteries. The events involved in a healthy pregnancy will then be compared to aberrant placentation and remodelling, which are characteristics of many pregnancy disorders, and recent advances in detection of abnormal placental development will also be discussed.

The phenotype of decidual CD56+ lymphocytes is influenced by secreted factors from decidual stromal cells but not macrophages in the first trimester of pregnancy.

During the first trimester of pregnancy the decidua is comprised of decidual stromal cells (DSC), invading fetal trophoblast cells and maternal leukocytes, including decidual natural killer (dNK) cells and macrophages. dNK cells are distinct from peripheral blood NK cells and have a role in regulating trophoblast invasion and spiral artery remodelling. The unique phenotype of dNK cells results from the decidual environment in which they reside, however the interaction and influence of other cells in the decidua on dNK phenotype is unknown. We isolated first trimester DSC and decidual macrophages and investigated the effect that DSC and decidual macrophage secreted factors have on CD56+ decidual lymphocyte receptor expression and cytokine secretion (including dNK cells). We report that DSC secreted factors induce the secretion of the cytokines IL-8 and IL-6 from first trimester CD56+ cells. However, neither DSC nor decidual macrophage secreted factors changed CD56+ cell receptor expression. These results suggest that secreted factors from DSC influence CD56+ decidual lymphocytes during the first trimester of pregnancy and therefore may play a role in regulating the unique phenotype and function of dNK cells during placentation.

Trophoblast-mediated spiral artery remodelling: a role for apoptosis.

In the first 20 weeks of pregnancy a number of important changes take place in the maternal uterine vasculature. Vascular endothelial and smooth muscle cells are lost from the spiral arteries and are replaced by fetal trophoblast cells. The resulting increase in blood flow to the intervillous space ensures that the fetus receives the nutrients and respiratory gases required for growth. Failure of the vessels to remodel sufficiently is a common feature of pregnancy pathologies such as early pregnancy loss, intrauterine growth restriction and pre-eclampsia. Although there is evidence to suggest that some vascular changes occur prior to trophoblast invasion, it is clear that in the absence of trophoblast invasion the remodelling of the spiral arteries is reduced. The cellular and molecular mechanisms by which trophoblasts influence vessel structure have been little studied. Trophoblasts synthesize and release a plethora of cytokines and growth factors including members of the tumour necrosis factor family such as tumour necrosis factor alpha, Fas-ligand and tumour necrosis factor-related apoptosis-inducing ligand. Recent studies suggest that these factors may be important in regulating the remodelling process by inducing both endothelial cell and vascular smooth muscle cell apoptosis.

Soluble HLA-G regulates motility and invasion of the trophoblast-derived cell line SGHPL-4.

BACKGROUND: Soluble human leucocyte antigen-G (sHLA-G) is secreted by extravillous trophoblast (EVT) and has roles in regulating immune cells within the decidua. HLA-G expression on EVT increases as they approach uterine spiral arteries and we have suggested that sHLA-G may be important in the remodelling of these vessels. The autocrine role of sHLA-G in regulating trophoblast function at this critical phase has not been studied. We aimed to investigate the effects of sHLA-G on trophoblast motility, invasion and survival. METHODS: The human EVT line, SGHPL-4, was stably transfected to over-express sHLA-G (SGHPL-4sG1). Motility and apoptosis were assessed by time-lapse microscopy. Cells were cultured on microcarrier beads embedded in fibrin gels to assess invasion. The effect of sHLA-G expression on motility, invasion and apoptosis in response to stimulation with either hepatocyte growth factor (HGF) or epidermal growth factor (EGF) was determined. RESULTS: There was no difference in the motility of either SGHPL-4 cells or SGHPL-4sG1 cells in the absence of stimulation. However, sHLA-G inhibited HGF-induced EVT motility. HGF- and EGF-induced invasions were significantly inhibited in SGHPL-4sG1 compared with SGHPL-4 cells. Increased expression of HLA-G had no significant effect on tumour necrosis factor (TNF)-alpha/actinomycin-induced apoptosis. CONCLUSIONS: Growth factor-stimulated trophoblast motility and invasion are regulated by sHLA-G, indicating a novel autocrine role. The inhibition of trophoblast invasion at the spiral artery may be important to allow interactions leading to vascular remodelling.

Fetal-derived trophoblast use the apoptotic cytokine tumor necrosis factor-alpha-related apoptosis-inducing ligand to induce smooth muscle cell death.

Remodeling of the uterine spiral arteries during pregnancy transforms them from high to low resistance vessels that lack vasoconstrictive properties. This process is essential to meet the demand for increased blood flow imposed by the growing fetus. Loss of endothelial and smooth muscle cells (SMC) is evident in remodeled arteries but the mechanisms underlying this transformation remain unknown. This study investigated the hypothesis that fetal trophoblast invading from the placenta instigate remodeling by triggering cell death in vascular SMC. Specifically, a role for trophoblast-derived death inducing cytokine tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) was investigated. Expression of the activating TRAIL receptors R1 and R2 was detected by flow cytometry on human aortic SMC and by immunohistochemistry on spiral artery SMC. Recombinant human TRAIL induced human aortic SMC apoptosis, which was inhibited by antibodies against TRAIL-R1 or -R2. Perfusion of denuded spiral artery segments with recombinant human TRAIL also induced SMC apoptosis. Trophoblasts isolated from first trimester placenta expressed membrane-associated TRAIL and induced apoptosis of human aortic SMC; apoptosis was significantly inhibited by a recombinant human TRAIL-R1:Fc construct. Trophoblast within the first trimester placental bed also expressed TRAIL. These data show that: 1) TRAIL causes SMC death; 2) trophoblast produce the apoptotic cytokine TRAIL; and 3) trophoblast induce SMC apoptosis via a TRAIL-dependent mechanism. We conclude that TRAIL produced by trophoblast causes apoptosis of SMC and thus may contribute to SMC loss during spiral artery remodeling in pregnancy.

The androgen and progesterone receptors regulate distinct gene networks and cellular functions in decidualizing endometrium.

Progesterone is indispensable for differentiation of human endometrial stromal cells (HESCs) into decidual cells, a process that critically controls embryo implantation. We now show an important role for androgen receptor (AR) signaling in this differentiation process. Decreased posttranslational modification of the AR by small ubiquitin-like modifier (SUMO)-1 in decidualizing cells accounted for increased responsiveness to androgen. By combining small interfering RNA technology with genome-wide expression profiling, we found that AR and progesterone receptor (PR) regulate the expression of distinct decidual gene networks. Ingenuity pathway analysis implicated a preponderance of AR-induced genes in cytoskeletal organization and cell motility, whereas analysis of AR-repressed genes suggested involvement in cell cycle regulation. Functionally, AR depletion prevented differentiation-dependent stress fiber formation and promoted motility and proliferation of decidualizing cells. In comparison, PR depletion perturbed the expression of many more genes, underscoring the importance of this nuclear receptor in diverse cellular functions. However, several PR-dependent genes encode for signaling intermediates, and knockdown of PR, but not AR, compromised activation of WNT/beta-catenin, TGFbeta/SMAD, and signal transducer and activator of transcription (STAT) pathways in decidualizing cells. Thus, the nonredundant function of the AR in decidualizing HESCs, centered on cytoskeletal organization and cell cycle regulation, implies an important role for androgens in modulating fetal-maternal interactions. Moreover, we show that PR regulates HESC differentiation, at least in part, by reprogramming growth factor and cytokine signal transduction.