Decidual cell regulation of trophoblast is altered in pregnancies at risk of pre-eclampsia.

Successful implantation and placentation are dependent on the interaction between decidual stromal cells (DSC) and extravillous trophoblast (EVT) cells. The extent of trophoblast invasion relies on communication between the placenta and maternal decidua. The cyclical process of decidualisation induces a transformation of endometrial fibroblasts to secretory DSC; these secreted products have many functions including the control of trophoblast invasion. Inadequate trophoblast invasion and remodelling of the uterine vessels (the spiral arteries) are associated with pregnancy disorders such as pre-eclampsia. Uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure of remodelling. DSC were isolated from pregnancies with normal (normal RI) or impaired (high RI) spiral artery remodelling. Following isolation, DSC were re-decidualised using cAMP and MPA and secretion of the decidualisation markers IGFBP-1 and prolactin assessed. We examined the impact of DSC-secreted factors on trophoblast cell function, using the EVT cell line SGHPL-4. We demonstrated that DSC exposed to decidual factors were able to re-decidualise in vitro and that the chemoattraction of trophoblasts by DSC is impaired in pregnancies with high RI. This study provides new insights into the role that DSC play in regulating EVT functions during the first trimester of pregnancy. This is the first study to demonstrate that DSC from pregnancies with impaired vascular remodelling in the first trimester secrete factors that inhibit the directional movement of trophoblast cells. This finding may be important in understanding aberrant trophoblast invasion in pregnancies where vascular remodelling is impaired.

Macrophage polarisation affects their regulation of trophoblast behaviour.

INTRODUCTION: During the first trimester of human pregnancy, fetally-derived extravillous trophoblast (EVT) invade into the uterine decidua and remodel the uterine spiral arteries to ensure that sufficient blood reaches the maternal-fetal interface. Decidual macrophages have been implicated in the regulation of decidual remodelling, and aberrant activation of these immune cells is associated with pre-eclampsia. METHODS: The monocytic cell line THP-1 was activated to induce a classically- or alternatively-activated macrophage phenotype and the conditioned media was used to treat the EVT cell line SGHPL-4 in order to determine the effect of macrophage polarisation on trophoblast behaviour in-vitro. SGHPL-4 cell functions were assessed using time-lapse microscopy, endothelial-like tube formation assays, and western blot. RESULTS: The polarisation state of the THP-1 cells was found to differentially alter the behaviour of trophoblast cells in-vitro with pro-inflammatory classically-activated macrophage conditioned media significantly inhibiting trophoblast motility, impeding trophoblast tube formation, and inducing trophoblast expression of cleaved caspase 3, when compared to anti-inflammatory alternatively-activated macrophage conditioned media. DISCUSSION: Macrophages can regulate trophoblast functions that are critical during decidual remodelling in early pregnancy. Importantly, there is differential regulation of trophoblast function in response to the polarisation state of these cells. Our studies indicate that the balance between a pro- and anti-inflammatory environment is important in regulating the cellular interactions at the maternal-fetal interface and that disturbances in this balance likely contribute to pregnancy disorders associated with poor trophoblast invasion and vessel remodelling.

Inhibition of DDAH1, but not DDAH2, results in apoptosis of a human trophoblast cell line in response to TRAIL.

STUDY QUESTION: Does inhibition of dimethylarginine dimethylaminohydrolase (DDAH) increase the sensitivity of trophoblasts to TRAIL-induced apoptosis? SUMMARY ANSWER: Inhibition of DDAH1, but not DDAH2, increases the sensitivity of trophoblasts to TRAIL-induced apoptosis. WHAT IS KNOWN ALREADY: Successful human pregnancy is dependent on adequate trophoblast invasion and remodelling of the maternal spiral arteries. Increased trophoblast apoptosis is seen in pregnancies complicated by pre-eclampsia. The mechanism underlying this increase is unknown. We have previously shown that nitric oxide (NO) is involved in regulating trophoblast motility and invasion, and have also demonstrated an important role for NO in regulating trophoblast sensitivity to apoptotic stimuli. DDAH is an enzyme that metabolizes asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis, previously shown to be elevated in the plasma of pre-eclamptic mothers. STUDY DESIGN, SIZE, DURATION: This study used the human extravillous trophoblast-derived cell line SGHPL-4 cells. All experiments were performed at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effect of DDAH on trophoblast apoptosis was examined using siRNA and time-lapse microscopy. Changes in the expression of DDAH were followed by PCR and western blot analysis. Receptor expression was followed by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Inhibiting the expression of DDAH1, but not DDAH2, resulted in a significant increase in the sensitivity of the EVT cell line SGHPL-4 to tumour necrosis factor related apoptosis inducing ligand (TRAIL) induced apoptosis (P < 0.01). This response could be mimicked by the addition of Asymmetric Dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis and the substrate for both isoforms of DDAH. We further showed that this increased sensitivity to apoptosis is accompanied by a significant increase in the expression of TRAIL receptor 2 (TR2; P < 0.05) but not TRAIL receptor 1 (TR1). LIMITATIONS, REASONS FOR CAUTION: This study was performed only in vitro using a well characterized trophoblast cell line, SGHPL-4, derived from first trimester extravillous trophoblasts. WIDER IMPLICATIONS OF THE FINDINGS: This study provides new insight into the role of the DDAH/ADMA pathway in the regulation of trophoblast function. Both dysregulation of DDAH and the accumulation of ADMA have been associated with the development of pre-eclampsia. This is the first study to implicate the DDAH/ADMA pathway as a mechanism that might underlie the poor trophoblast invasion seen in this common pregnancy disorder. STUDY FUNDING/COMPETING INTERESTS: B.A.L. was supported by a grant from Action Medical Research UK (SP4577). A.E.W. was supported by a grant from the Wellcome Trust (091550). There are no competing interests and the authors have no conflict interest to declare.

Expression of voltage-dependent potassium channels in first trimester human placentae.

Potassium channel α-subunits encoded by KCNQ1-5 genes form voltage-dependent channels (KV7), modulated by KCNE1-5 encoded accessory proteins. The aim was to determine KCNQ and KCNE mRNA expression and assess protein expression/localisation of the KCNQ3 and KCNE5 isoforms in first trimester placental tissue. Placentae were obtained from women undergoing elective surgical termination of pregnancy (TOP) at ≤ 10 weeks' (early TOP) and >10 weeks' (mid TOP) gestations. KCNQ1-5 expression was unchanged during the first trimester. KCNE5 expression increased in mid TOP vs. early TOP samples (P = 0.022). This novel study reports mRNA and protein expression of KV7 channels in first trimester placentae.

Increased angiogenic factor secretion by decidual natural killer cells from pregnancies with high uterine artery resistance alters trophoblast function.

STUDY QUESTION: Are the concentrations of factors secreted by decidual natural killer (dNK) cells from pregnancies at high risk of poor spiral artery remodelling different to those secreted from pregnancies at low risk? SUMMARY ANSWER: Expression levels of PLGF, sIL-2R, endostatin and angiogenin were significantly increased by dNK cells from high-risk pregnancies, and angiogenin and endostatin were found to alter trophoblast function. WHAT IS KNOWN ALREADY: During early pregnancy, maternal uterine spiral arteries are remodelled from small diameter, low-flow, high-resistance vessels into larger diameter, higher flow vessels, with low-resistance. This change is essential for the developing fetus to obtain sufficient oxygen and nutrients. dNK cells have been implicated in this process. STUDY DESIGN, SIZE, DURATION: dNK cells were isolated from first trimester terminations of pregnancies (obtained with local ethical approval) screened for normal- or high-resistance index, indicative of cases least (<1%) and most (>21%) likely to have developed pre-eclampsia had the pregnancy not been terminated (n = 18 each group). Secreted factors and the effects of these on the trophoblast cell line, SGHPL-4, were assessed in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: A multiplex assay was used to assess dNK cell-secreted factors. SGHPL-4 cell functions were assessed using time-lapse microscopy, 3D invasion assays, endothelial-like tube formation ability and western blot analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The expression levels of PLGF (P < 0.01), sIL-2R (P < 0.01), endostatin (P < 0.05) and angiogenin (P < 0.05) were significantly increased by dNK cells from high-risk pregnancies. Endostatin significantly decreased SGHPL-4 invasion (P < 0.05), SGHPL-4 tube formation (P < 0.05) and SGHPL-4 Akt(ser473) phosphorylation (P < 0.05). Angiogenin significantly decreased SGHPL-4 invasion (P < 0.05), but increased SGHPL-4 tube formation (P < 0.01) and decreased SGHPL-4 Akt(ser473) phosphorylation (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The culture of dNK cells and protein concentrations in vitro may not fully represent the in vivo situation. Although SGHPL-4 cells are extravillous trophoblast derived, further studies would be needed to confirm the roles of angiogenin and endostatin in vivo. WIDER IMPLICATIONS OF THE FINDINGS: The altered expression of secreted factors of dNK cells may contribute to pregnancy disorders associated with poor spiral artery remodelling. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Wellcome Trust (project reference 091550). R.F. was a recipient of a PhD studentship from the Division of Biomedical Sciences, St. George's, University of London. The authors have no conflict of interests.

OS081. Novel KCNQ3/KCNE5 isoform protein and mRNA expression in first trimester human placentae.

INTRODUCTION: Potassium channel α-subunits encoded by KCNQ1-5 genes (Kv7) form voltage-dependent channels that can be modulated by KCNE1-5 encoded accessory proteins. These channels are known to play a role in the reactivity of blood vessels. We have previously shown that both mRNA and protein expression for the novel combination of KCNQ3 and KCNE5 are increased in term and preterm pre-eclampsia (PE) compared to normotensive control placentae [1]. The expression of these isoforms in early placental tissue has not been examined. OBJECTIVES: The aims of this study were to determine whether KCNQ3 and KCNE5 mRNA and proteins are expressed in first trimester placental tissue. METHODS: Placental samples were obtained from women undergoing elective surgical termination of pregnancy between 6 and 12 weeks' gestation (n=7) following informed written consent. KCNQ3 and KCNE5 mRNA expression was measured by qRT-PCR and normalised to stably expressed GAPDH. Immunohistochemistry was used to assess protein expression and localisation of the isoforms. RESULTS: Both mRNA and protein expression of KCNQ3 and KCNE5 were detected in placental tissue at all gestations. KCNE5 mRNA expression remained constant between 6 and 10 weeks with a subsequent rise at 11 and 12 weeks. KCNQ3 mRNA expression was initially lower than KCNE5 but markedly increased at 7 weeks remaining high until 10 weeks and falling below KCNE5 levels by 12 weeks. Protein expression for both KCNQ3 and KCNE5 was localised mainly to the syncytiotrophoblast but was also evident in the mesenchyme; overall KCNQ3 intensity significantly increased with gestational age (p=0.044). CONCLUSION: KCNQ3 and KCNE5 channel isoforms are highly expressed in first trimester placenta. The temporal changes in mRNA expression mirror changes in the placental tissue oxygen tension which increases between 8 and 10 weeks. This would precede the dislocation of the spiral artery plugs enabling maternal blood to flow freely and continuously into the intervillous spaces. We speculate that the increase in mesenchymal protein expression may be related to angiogenesis during this critical window of feto-placental vascular development. Future work will characterise the complete KCNQ/KCNE isoforms in first trimester placental tissue and assess potential functional roles of these channels both in early placentation and in relation to PE. FUNDING: Tommy's Charity (Registered charity 1060508).

Elevated glucocorticoid metabolism in placental tissue from first trimester pregnancies at increased risk of pre-eclampsia.

BACKGROUND: The local actions of glucocorticoids in the placenta can be modulated by 11ß-hydroxysteroid dehydrogenase (11ßHSD) enzymes, which catalyse inter-conversion of cortisol with its inert metabolite, cortisone, and are known to be expressed in the term placenta and decidua. However, the expression and activity of these enzymes have not been well characterised in the first trimester placenta. The aim of this study was to compare 11ßHSD2 expression and activity in first trimester placental tissue from pregnancies at either relatively low or high risk of developing pre-eclampsia as determined by Doppler ultrasound. METHODS: Enzyme expression was assessed by western blot analysis and immunohistochemistry while 11ßHSD enzyme activities were quantified using radiometric conversion of [3H]-cortisol in the presence of NADP(+) or NAD(+). RESULTS: 11ßHSD2 was expressed in syncytiotrophoblast of first trimester placenta, and there was no difference in the level of expression of placental 11ßHSD2 protein between 9 high pre-eclampsia risk and 14 low pre-eclampsia risk pregnancies. NAD(+)-dependent cortisol oxidation was elevated 3-fold in placental tissue from pregnancies at higher risk of pre-eclampsia than in normal pregnancies (50.9 ± 15.9 versus 18.3 ± 1.9 pmol cortisone/mg protein.10 min, n = 11 & 12, respectively; P < 0.05). CONCLUSIONS: Expression of 11ßHSD2 is thought to protect the fetus from exposure to maternal cortisol. While other studies have suggested that 11ßHSD2 is down regulated in term pre-eclamptic placentae, our study suggests that there is increased cortisol inactivation in first trimester placenta prior to week 10 of gestation, from pregnancies at higher risk of developing pre-eclampsia.

The role of tumour-derived iNOS in tumour progression and angiogenesis.

BACKGROUND: Progressive tumour growth is dependent on the development of a functional tumour vasculature and highly regulated by growth factors and cytokines. Nitric oxide (NO) is a free radical, produced both by tumour and host cells, and functions as a signalling molecule downstream of several angiogenic factors. Both pro- and antitumourigenic properties have been attributed to NO. METHODS: The expression of the inducible isoform of NO synthase (iNOS) was knocked down in the C6 glioma cell line using constitutive expression of antisense RNA, and the effect of tumour-derived NO on tumour progression and angiogenesis was investigated. RESULTS: Tumours in which iNOS expression was decreased displayed significantly reduced growth rates compared with tumours derived from parental C6 cells. Quantitative non-invasive magnetic resonance imaging and fluorescence microscopy of tumour uptake of Hoechst 33342, and haematoxylin and eosin staining, revealed significantly impaired vascular development and function in antisense iNOS tumours compared with control in vivo, primarily associated with the more necrotic tumour core. Decreased iNOS expression had no effect on tumour VEGF expression. CONCLUSION: Nitric oxide derived from tumour iNOS is an important modulator of tumour progression and angiogenesis in C6 gliomas and further supports the therapeutic strategy of inhibiting iNOS for the treatment of cancer.

Cellular and molecular regulation of spiral artery remodelling: lessons from the cardiovascular field.

A number of important changes take place in the maternal uterine vasculature during the first few weeks of pregnancy resulting in increased blood flow to the intervillous space. Vascular endothelial and smooth muscle cells are lost from the spiral arteries and are replaced by fetal trophoblast cells. 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. There is evidence to suggest that some vascular changes occur prior to trophoblast invasion, however, in the absence of trophoblasts remodelling of the spiral arteries is reduced. Until recently our knowledge of these events has been obtained from immunohistochemical studies which, although extremely useful, can give little insight into the mechanisms involved. With the development of more complex in vitro models a picture of events at a cellular and molecular level is beginning to emerge, although some caution is required in extrapolating to the in vivo situation. Trophoblasts synthesise and release a plethora of cytokines and growth factors including members of the tumour necrosis factor family. Studies suggest that these factors may be important in regulating the remodelling process by inducing both endothelial and vascular smooth muscle cell apoptosis. In addition, it is evident from studies in other vascular beds that the structure of the vessel is influenced by factors such as flow, changes in the composition of the extracellular matrix, the phenotype of the vascular cells and the local immune cell environment. It is the aim of this review to present our current knowledge of the mechanisms involved in spiral artery remodelling and explore other possible pathways and cellular interactions that may be involved, informed by studies in the cardiovascular field.

Live cell image analysis of cell-cell interactions reveals the specific targeting of vascular smooth muscle cells by fetal trophoblasts.

In early pregnancy, fetal trophoblasts selectively invade and remodel maternal spiral arteries. A healthy pregnancy is dependent on this adaptation to allow sufficient maternal blood to reach the placenta and the developing fetus. However, little is known of the role played by trophoblasts in this adaptation process. In this study, the interactions between trophoblast cells (TC) and vascular smooth muscle cells (VSMC) were examined using novel live cell image analysis methods which allow quantitative assessment of the behaviour of these two cell types in co-culture. TC and VSMC were simultaneously tracked in co-culture and, for each cell type, directionality, speed and the cell-cell interaction were assessed. The overall migratory behaviour of TC was markedly different in the presence of VSMC with co-cultured TC migrating further with directional movement while mono-cultured TC moved more randomly. Furthermore, TC were shown to specifically target VSMC, suggesting that invading TC may initiate targeted vascular remodelling. Analysis of movement behaviour and cell-cell attraction will be useful in other co-culture systems in addition to answering important questions in the reproductive field.

BeWo cells stimulate smooth muscle cell apoptosis and elastin breakdown in a model of spiral artery transformation.

BACKGROUND: During pregnancy, extravillous trophoblast invades the uterine wall and enters the spiral arteries. Remodelling ensues, with loss of vascular smooth muscle cells (SMCs) to create high flow, low resistance vessels. Pregnancies complicated by pre-eclampsia are characterized by incomplete arterial remodelling. Endovascular trophoblast is not easily accessible for studies to establish the pathogenesis of pre-eclampsia, so we have developed a model appropriate to carry out mechanistic studies of vessel wall transformation. METHODS AND RESULTS: Segments of human spiral artery were perfused with the choriocarcinoma cell line, BeWo; cells invaded the vessel wall and induced apoptosis of vascular SMC. Perfusion of vessels with BeWo-conditioned medium also induced SMC apoptosis, indicating the presence of a soluble apoptotic factor. BeWo express Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment of BeWo-conditioned medium with antibodies against FasL inhibited vascular SMC apoptosis in vitro. Antibodies that blocked TRAIL receptor function had no effect. Extracellular matrix degradation is also a prerequisite for vascular remodelling; BeWo express matrix metalloproteinase-12 (MMP-12) and BeWo-conditioned medium increased MMP-12 expression in spiral artery SMC. CONCLUSIONS: BeWo induce arterial remodelling via FasL- and MMP-12-dependent mechanisms. BeWo-derived factors up-regulate protease expression in spiral artery SMC to facilitate matrix breakdown.

Effect of heparin and fractionated heparin on trophoblast invasion.

BACKGROUND: Heparin can significantly reduce pregnancy complications in women with certain thrombophilias, such as antiphospholipid syndrome. Recent reports suggest that heparin may act by mechanisms other than anticoagulation. However, the effect of heparin on trophoblast biology in the absence of thrombophilia has not been extensively investigated. Therefore, this study aimed to evaluate trophoblast invasion, using an established cell line and primary extravillous trophoblasts (EVTs), following exposure to heparin and fractionated heparin. METHODS: An EVT cell line (SGHPL-4) was used to study invasion in the presence of hepatocyte growth factor (HGF) and varying concentrations of fractionated and unfractionated heparin. These experiments were repeated using first trimester primary EVTs. RESULTS: Both forms of heparin significantly reduced HGF-induced invasion in the SGHPL-4 cell line. This suppression of invasion appeared to be dose-dependent for fractionated heparin. In primary EVT cells, fractionated heparin also demonstrated significant suppression of invasion. CONCLUSIONS: Heparin has the potential to reduce trophoblast invasion in cell lines and first trimester EVT cells. This article highlights the need for further evaluation of these medications in vitro and in vivo, especially when used in the absence of thrombophilic disorders.

Dimethylarginine dimethylaminohydrolase (DDAH) regulates trophoblast invasion and motility through effects on nitric oxide.

BACKGROUND: Invasion of trophoblast into the uterine environment is crucial for establishing a successful pregnancy. Physiological production of nitric oxide (NO) by extravillous trophoblasts results in significant pro-invasive effects. NO synthesis is competitively inhibited by methylated arginine analogues such as asymmetric dimethylarginine (ADMA) but not the enantiomer symmetric dimethylarginine (SDMA). Within cells, the concentration of ADMA is regulated by the activity of the enzyme dimethylarginine dimethylaminohydrolase (DDAH). The aim of this study was to examine DDAH expression and function in trophoblasts. METHODS AND RESULTS: DDAH-1 and DDAH-2 messenger RNA and protein were demonstrated in first trimester placental tissue, primary extravillous trophoblasts and extravillous trophoblast-derived cell lines. DDAH activity was demonstrated in both cells and tissue. Overexpression of DDAH-1 in trophoblasts led to a number of significant changes, including an 8-fold increase in enzymatic activity, a 59% decrease in production of ADMA (but not SDMA), a 1.9-fold increase in NO and a 1.6-fold increase in cyclic guanosine monophosphate (cGMP) production. Functional assays showed that increased DDAH activity led to significantly increased cell motility and invasion in response to hepatocyte growth factor (HGF). CONCLUSIONS: DDAH may play an important role in the regulation of extravillous trophoblast function via its effects on ADMA and NO production.

Effect of first-trimester serum from pregnant women with high-resistance uterine artery Doppler resistance on extravillous trophoblast invasion.

BACKGROUND: Abnormal uterine artery Doppler indices are associated with pregnancy complications such as pre-eclampsia and intrauterine growth restriction. Poor trophoblast invasion may be a consequence of, or be associated with, abnormal Doppler indices. OBJECTIVE: To evaluate in vitro trophoblast function following exposure to first-trimester serum from pregnancies with high uterine artery Doppler resistance indices. METHODS: Doppler ultrasound examination of the maternal uterine arteries was performed on women at 10-14 weeks' gestation. Serum was collected from women with bilateral uterine artery notches with resistance indices above the 95th centile and from patients with normal uterine artery indices. The effect of serum on trophoblast invasion was determined using an established in vitro model from the extravillous trophoblast-derived cell line SGHPL-4. RESULTS: Trophoblastic invasion was significantly reduced when treated with serum from women with high-resistance compared with normal-resistance uterine artery Doppler indices (P < 0.05). CONCLUSION: Maternal serum in the first trimester of pregnancy from patients with high-resistance uterine artery Doppler indices appears to inhibit trophoblast invasion. This experimental model allows further investigation of factors responsible and the evaluation of therapeutic strategies.

Trophoblast interactions with endothelial cells are increased by interleukin-1beta and tumour necrosis factor alpha and involve vascular cell adhesion molecule-1 and alpha4beta1.

Interactions between fetal extravillous trophoblast cells and maternal uterine cells are of critical importance in successful placentation. In the first trimester, trophoblasts invade the uterine environment and reach the spiral arteries where they interact with vascular cells; however, little is known of the nature of these interactions. We have developed a fluorescent binding assay to investigate the contact between trophoblasts and endothelial cells and to determine its regulation by cytokines and adhesion molecules. Stimulation of an endothelial cell line (SGHEC-7) with interleukin-1beta or tumour necrosis factor-alpha significantly increased adhesion of the first-trimester extravillous trophoblast-derived cell line, SGHPL-4. Using blocking antibodies, vascular cell adhesion molecule-1 (VCAM-1) and integrin alpha4beta1 (VLA-4), but not intercellular adhesion molecule-1 (ICAM-1), were shown to be important in trophoblast binding to activated endothelial cells. SGHPL-4 cells were shown to express HLA-G, alpha4beta1 and ICAM-1 at high levels and LFA-1 and VCAM-1 at lower levels. ICAM-1 and VCAM-1 are expressed on SGHEC-7 cells and their expression was confirmed on primary decidual endothelial cells. In conclusion, we have demonstrated the importance of VCAM-1 and alpha4beta1 in trophoblasts-endothelial interactions. Improved knowledge of the nature of these fetal-maternal interactions will have implications for understanding situations when placentation is compromised.

Transforming growth factor-beta1 regulates hepatocyte growth factor-induced trophoblast motility and invasion.

During placental development extravillous trophoblasts invade the uterine wall in a tightly regulated manner dependent on both pro- and anti-invasive molecules. We have shown using the extravillous trophoblast cell line, SGHPL-4, that both cellular invasion and motility are stimulated by hepatocyte growth factor (HGF). It has previously been demonstrated that transforming growth factor=beta1 (TGF-beta1), produced by the decidua, inhibits extravillous trophoblast proliferation and invasion. It was the aim of this study to determine whether TGF-beta1 could modulate HGF-induced motility and invasion and, if so, examine the mechanism involved. TGF-beta1 significantly inhibited the growth of SGHPL-4 cells stimulated with 10 per cent serum. HGF-stimulated trophoblast cell invasion and motility were significantly inhibited by TGF-beta1. Neither HGF nor TGF-beta1 had an effect on SGHPL-4 cell growth under the conditions used for the invasion and motility experiments (0.5 per cent serum). Previous studies suggest that both HGF-stimulated trophoblast invasion and motility may be regulated by the production of nitric oxide. TGF-beta1 was found to significantly decrease HGF-induced iNOS expression therefore suggesting a novel mechanism by which TGF-beta1 could regulate motility and invasion.

Dimethylarginine dimethylaminohydrolase I enhances tumour growth and angiogenesis.

Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase I. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis in vitro. Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development. Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production.

Trophoblast invasion of spiral arteries: a novel in vitro model.

Extravillous trophoblasts invade the uterine wall (interstitial invasion) and the spiral arteries (endovascular invasion), replacing the cells of the vessel wall and creating a high-flow low-resistance vessel. We have developed a novel model to allow the interactions between the invading trophoblast cells and the cells of the spiral artery to be directly examined. Unmodified (non-placental bed) spiral arteries were obtained from uterine biopsies at caesarean section. Fluorescently labelled trophoblasts were seeded on top of artery segments embedded in fibrin gels (to study interstitial invasion) or perfused into the lumen of arteries mounted on a pressure myograph (to study endovascular invasion). Trophoblasts were incubated with the vessels for 3-5 days prior to cryo-sectioning. Both interstitial and endovascular interactions/invasion could clearly be detected and a comparison of the extravillous trophoblast cell line, SGHPL-4 and primary first trimester cytotrophoblasts showed both to be invasive in this model. This novel method will prove useful in an area where in vitro studies have been hampered by the lack of suitable models directly examining cellular interactions during invasion.