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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.

What are the effects of the eotaxin group of chemokines (CCL11, CCL24 and CCL26) on extravillous trophoblast (EVT) functions important during uterine decidual vessel remodelling?CCL11, CCL24 and CCL26 can regulate EVT migration, invasion and adhesion, highlighting a potential regulatory role for these chemokines during uterine decidual spiral arteriole remodelling in the first trimester of human pregnancy.A successful human pregnancy depends on adequate remodelling of the uterine decidual spiral arterioles, a process carried out by EVT which invade from the placenta. The invasion by EVT into the maternal uterine decidual vessels is regulated by the interaction of many factors including members of the chemokine subfamily of cytokines.This study used the HTR8/SVneo cell line as a model for invasive EVT. All experiments were repeated on at least three separate occasions.The effect of recombinant human CCL11, CCL24 and CCL26 on EVT migration and invasive potential was measured using the xCELLigence real-time system, wound-healing and Matrigel invasion assays, zymography to measure MMP activity and reverse zymography to measure TIMP activity. A commercially available adhesion assay was used to assess EVT adhesion to extracellular matrix proteins.All the three eotaxins were found to significantly stimulate migration of the EVT-derived cell line HTR8/SVneo (P0.05) with no significant changes in cell number following treatment with each chemokine (P0.05). All the three eotaxins significantly increased HTR8/SVneo invasion (P0.05) and MMP2 activity (P0.05) without any effects on TIMP2 activity (P0.05). All the three eotaxins significantly increased HTR8/SVneo cell binding to collagen IV (P0.05) and fibronectin (P0.05).This work has been conducted in vitro with a commonly used cell line model of EVT, HTR8/SVneo.This study is the first to comprehensively examine the effects of the eotaxin group of chemokines on EVT functions and demonstrates that all the three eotaxins have the ability to regulate EVT functions critical to their role in vessel remodelling. This identifies a new role for the eotaxin group of chemokines during placentation.

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.

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-α–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.

Objective We investigated the hypothesis that antiphospholipid antibodies (aPL) have a detrimental effect on human extravillous trophoblast (EVT) differentiation into giant multinucleated cells "in vitro." Design The EVT were isolated from the placental chorion using enzymatic digestion and Percoll gradient centrifugation. After 24, 36, and 48 hours in culture, giant multinuclear cells (GMC) were identified by immunohistochemistry using antibodies to cytokeratin 7 and counted. Setting An academic research laboratory. Patient(s) Placentas were donated by women having an elective cesarean section for a normal pregnancy at term. Main outcome measure(s) This model was then used to investigate the effect of two different monoclonal aPL to β 2 -glycoprotein 1 (IIC5 and ID2), and control mouse IgG antibody on EVT differentiation. Result(s) Freshly isolated EVT were nonproliferative but moved together losing their intervening cell walls and differentiated into GMC. Maximal numbers of GMC were detected after 48 hours of culture. The aPL, IIC5, and ID2 significantly inhibited GMC formation, whereas the mouse IgG control had no effect. Conclusion(s) Antiphospholipid antibodies can inhibit EVT differentiation and GMC formation "in vitro" suggesting that a failure of trophoblast differentiation and subsequent uteroplacental development may be an underlying pathology in antiphospholipid syndrome-associated pregnancy loss.

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.

all- trans -Retinoic acid (atRA) has important effects on the developing and mature cardiovascular system. Nitric oxide (NO) production has been associated with the atRA-induced differentiation of neuronal cells, and we hypothesized that NO may also mediate certain actions of atRA in the cardiovascular system. We studied the effects of atRA on NO production by endothelial cells and determined whether regulation of enzymes responsible for metabolism of asymmetric dimethylarginine (ADMA) contributed to the effects seen. Murine endothelioma (sEnd.1) cells were incubated with or without atRA. Nitrite production was determined using the Griess reaction. The expression of NO synthase (NOS) and dimethylarginine dimethylaminohydrolase (DDAH) genes was determined by Northern blotting. A reporter gene assay was also used to study the effect of atRA on the DDAH II promoter. atRA significantly increased nitrite production by sEnd.1 cells despite no increase in eNOS expression. atRA also increased DDAH II gene expression and promoter activity and reduced the ratio of ADMA to symmetric dimethylarginine (SDMA) in culture medium. The DDAH inhibitor 4124W significantly reduced the induction of NO synthesis by atRA. The present study demonstrates that atRA increases NO synthesis in endothelial cells without increasing eNOS expression. atRA also increases the expression of DDAH II, the predominant DDAH isoform in endothelial cells. Our data suggests that the induction of NO synthesis by atRA may be facilitated by DDAH II. This pathway may help to explain some of the effects of atRA on the cardiovascular system.

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.

Proliferation of endothelial cells is a vital component of vascular repair and angiogenesis. The endothelial cell mediator, nitric oxide (NO) has been reported both to inhibit and to promote endothelial cell proliferation. In this study we have generated cell lines which constitutively express antisense RNA to a region of inducible nitric oxide synthase (iNOS) from a murine endothelial cell line, sEnd-1. In response to stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) these antisense cells had no detectable RNA for endogenous iNOS, barely detectable iNOS protein and produced 82% less NO than did the control transfected line. Stimulation of the control transfected line caused significant NO production and inhibition of cell growth whereas for the antisense line, producing little NO in response to stimulation, proliferation remained the same as for unstimulated cells. No differences in cell death were observed between unstimulated and LPS/IFN-gamma stimulated cells. The data presented in this study directly demonstrate that NO derived endogenously from iNOS inhibits proliferation of endothelial cells. This approach overcomes problems in other studies where NO donors or non-isoform specific inhibitors of NO synthase have been used.

NG,NG-dimethylarginine is an endogenous inhibitor of nitric oxide synthesis produced by endothelial cells and found in the plasma and urine of normal adults. We have examined the ability of NG, NG-dimethylarginine, produced by endothelial cells (SGHEC-7), to regulate the production of nitric oxide by lipopolysaccharide-stimulated mouse macrophage cells (J774.2). Stimulation of SGHEC-7 or J774.2 cells with lipopolysaccharide had no effect on their release of NG,NG-dimethylarginine into the culture supernatant. Stimulation of J774.2 cells with lipopolysaccharide for 24 h significantly stimulated nitric oxide production by J774.2 but not SGHEC-7 cells. When lipopolysaccharide-stimulated J774.2 cells were co-cultured with endothelial cells for 24 h, there was a significant inhibition of nitrite accumulation. The inhibition observed was dependent on the endothelial cell number (12 +/- 5% [mean +/- SEM] following incubation with 0.6 x 105 cells, up to 47 +/- 8% with 4.8 x 105 cells). The inhibitory effect of endothelial cells was prevented by incubation with increasing concentrations of L-arginine; the IC50 was 2.9 +/- 0.6 mM arginine. Western blot analysis indicated that the expression of inducible nitric oxide synthase was not inhibited by co-culture with SGHEC-7 cells. The results presented here demonstrate that NG,NG-dimethylarginine synthesized by endothelial cells may inhibit nitric oxide synthase in adjacent cells and play a role in the regulation of nitric oxide synthesis by macrophages.

1. The expression of hepatocyte growth factor (HGF) is essential for normal placental development although its function is unknown. In this study we examined the effect of HGF on trophoblast cell motility and invasion of fibrin gels and investigated the possible role of nitric oxide (NO) in this process. 2. The human extravillous trophoblast cell line SGHPL-4 express both the constitutive and inducible isoforms of nitric oxide synthase (NOS). 3. HGF significantly stimulates cell motility in monolayer culture, the invasion of fibrin gels and the production of guanosine 3':5'-cyclic monophosphate (cyclic GMP). 4. Invasion, motility and cyclic GMP production were inhibited by Ng-monomethyl-L-arginine (L-NMMA). 5. Cell motility was also significantly inhibited by the inducible NOS specific inhibitor 1400 W. 6. Neither 8 Br-cyclic GMP nor the NO donor spermine-NO had any significant effect on basal trophoblast cell motility. 7. The data presented in this study demonstrate a direct effect of trophoblast-derived NO synthesis on trophoblast cell function and support the idea that HGF is involved in the regulation of trophoblast invasion through mechanisms that involve the production of NO. However neither exogenous NO nor activation of cyclic GMP-dependent pathways alone are sufficient to stimulate trophoblast cell motility.

Recent studies have demonstrated the production of the multi-functional messenger molecule nitric oxide (NO) by the thyroid gland. To examine a possible role for NO in thyroid function, we studied the acute and chronic effect of NO donors on thyroid peroxidase (TPO) activity in monolayer cultures of primary human thyrocytes, using a colorimerric assay technique. The presence of either S-nitrosoglutathione (GS-NO) or sodium nitroprusside (SNP) (10 −6 –10 −4 M) at the time of the assay caused a significant increase in TPO activity. Pre-incubation of thyrocytes with 10 .5 M GS-NO for 3 days had no effect on the level of TPO activity when the assay was performed in the absence of NO donors. However, GS-NO pre-incubation significantly enhanced the acute stimulatory effect of GS-NO and SNP on TPO activity. These results suggest a possible role for NO in the regulation of TPO activity and thus thyroid hormone synthesis.

We investigated the change in the plasma concentration of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, in early-, mid-, and late-gestation normotensive pregnancies and in gestational age-matched preeclamptic pregnancies and compared the observed changes with changes in blood pressure.Blood pressure and peripheral plasma asymmetric dimethylarginine concentrations were measured in 20 nonpregnant and 145 pregnant women (33 first-trimester, 50 second-trimester, and 44 third-trimester normotensive pregnancies and 18 third-trimester pregnancies complicated by preeclampsia). In 23 normotensive pregnancies serial plasma asymmetric dimethylarginine concentrations were measured. Statistical analysis was by analysis of variance and linear regression.The blood pressures recorded throughout normal pregnancy were significantly lower than in nonpregnant subjects (p0.0001). The mean systolic, diastolic, and average blood pressures were significantly higher in the second-trimester groups than in the first-trimester groups, whereas in the third trimester average and diastolic blood pressures were significantly higher than in the second trimester. The mean (+/-SD) systolic and diastolic blood pressures in third-trimester preeclamptic patients was 157.7 +/- 11.2 and 110.9 +/- 8.5 mm Hg. The mean plasma asymmetric dimethylarginine concentration in nonpregnant women was 0.82 +/- 0.31 micromol/L (significantly higher than in normotensive pregnancy, p0.0001). The plasma asymmetric dimethylarginine concentration was also significantly higher in second-trimester than in first-trimester normotensive groups (respectively, 0.52 +/- 0.20 micromol/L and 0.40 +/- 0.15 micromol/L, p = 0.001) and was higher in third-trimester normotensive pregnancy 0.56 +/- 0.23 micromol/L than it was in the second trimester. The asymmetric dimethylarginine concentration in third-trimester preeclamptic patients was 1.17 +/- 0.42 micromol/L (p0.0001 vs normotensive third-trimester subjects).It is well recognized that blood pressure falls in early normal pregnancy and rises again toward term. These studies show that the early fall in blood pressure is accompanied by a significant fall in the plasma asymmetric dimethylarginine concentration. Later in pregnancy circulating concentrations increase and, when pregnancy is complicated by preeclampsia, concentrations are higher than in the nonpregnant state. Our data support a role for both asymmetric dimethylarginine and nitric oxide in the changes in blood pressure seen in both normal and preeclamptic pregnancy.

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 Conclusion— 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.

1. Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors NG-monomethyl-arginine and NG,NG-dimethy-L-arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates. 2. S-2-amino-4(3-methylguanidino)butanoic acid (4124W) inhibited the metabolism of [14C]-NG-monomethyl-L-arginine to [14C]-citrulline by rat liver homogenates (IC50 416 +/- 66 microM; n = 9), human cultured endothelial cells (IC50 250 +/- 34 microM; n = 9) and isolated purified dimethylarginine dimethylaminohydrolase. 3. Addition of 4124W to culture medium increased the accumulation of endogenously-generated NG,NG-dimethy-L-arginine in the supernatant of human cultured endothelial cells from 3.1 +/- 0.3 to 5 +/- 0.7 microM (n = 15; P < 0.005). 4. 4124W (1 microM - 1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium-dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 +/- 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L-arginine (100 microM). 4124W reversed endothelium-dependent relaxation of human saphenous vein (19.2 +/- 6.7% reversal of bradykinin-induced relaxation at 1 mM 4124W). 5. These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular contraction of NG,NG-dimethyl-L-arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.

The properties, selectivity, and regulation of NG-monomethyl-L-arginine (L-NMMA) uptake were examined in a human cultured vascular endothelial cell line SGHEC-7 and murine macrophage J774 cells. In both cell types the uptake of L-[14C]NMMA was time and temperature dependent. In endothelial cells L-[14C]NMMA uptake occurred via a single saturable carrier-mediated system with an apparent Kt of 77 +/- 2 microM. In murine macrophage cells a saturable component with an apparent Kt of 51 +/- 6 microM and a nonsaturable component of L-NMMA uptake were identified. In both cell types uptake of L-[14C]NMMA (10 microM) was significantly inhibited in the presence of 100-fold excess of L-NMMA, asymmetric NG,NG-dimethyl-L-arginine (ADMA), symmetric NG,NG-dimethyl-L-arginine (SDMA), L-canavanine, L-arginine, and to a lesser extent D-arginine. Uptake of L-[14C]NMMA was inhibited weakly (approximately 30%) by NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, aminoguanidine, and L-citrulline. Incubation of macrophage J774 cells with lipopolysaccharide (LPS; 1 or 10 micrograms/ml) resulted in the induction of nitric oxide (NO) synthase activity determined by the accumulation of nitrite in the culture medium. In these cells an enhanced uptake of L-NMMA uptake was observed which was prevented by pretreatment with cycloheximide (1 microM) but not dexamethasone (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

We report the characterization of a novel series of human endothelial cell lines (designated SGHEC) regarding the expression and release of adhesion molecules and their binding of lymphocytes. SGHEC expressed significant levels of intercellular adhesion molecule-1 (ICAM-1; CD54) which increased after stimulation with tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or interferon-gamma (IFN-gamma). Vascular cell adhesion molecule-1 (VCAM-1; CD106) and E-selectin (CD62E) were not detectable on unstimulated SGHEC but substantial levels were expressed after stimulation with either TNF alpha or IL-1 beta but not with IFN-gamma. The increased expression of ICAM-1 and VCAM-1 was evident after 4 h stimulation and was even higher after 24 h; E-selectin was maximal after 4 h and returned almost to basal levels by 24 h. Substantial quantities of immunoreactive ICAM-1 and VCAM-1 also accumulated as soluble material in the supernatants of TNF alpha-stimulated SGHEC (VCAM-1 was substantially higher than ICAM-1), but E-selectin remained below the limits of detection. Various quantitative data suggest that this is a controlled release regulated by cytokine and provide support for a physiological function for these soluble molecules. Primary human lymphocytes and lymphoblastoid cell lines expressing lymphocyte function-associated antigen-1 (LFA-1) bound to SGHEC; this binding increased substantially after activation of either cell type. The binding was inhibited by monoclonal antibodies against LFA-1 and, to a lesser extent, ICAM-1, thus demonstrating the importance of these molecules in the observed binding; neither anti-VCAM-1 nor anti-E-selectin antibodies affected the binding. From these various data, we conclude that LFA-1/ICAM-1 interactions are partially responsible for the binding of lymphocytes to endothelial cells. The SGHEC lines should prove useful in investigating leukocyte-endothelial interactions and the mechanism of release of soluble adhesion molecules.

1. The metabolism of methylarginines by human cultured endothelial cells and human saphenous vein was studied in vitro. The human endothelial cell line (SGHEC-7), primary cultures of human umbilical vein endothelial cells (HUVEC) and human saphenous vein were incubated with [14C]-monomethyl-L-arginine ([14C]-L-NMMA) and the cytosolic extract analysed by high performance liquid chromatography (h.p.l.c.) with on-line radioisotope detection. 2. SGHEC-7, HUVEC and human saphenous vein metabolized [14C]-L-NMMA to a compound which co-eluted with [14C]-citrulline. A second metabolite which co-eluted with [14C]-arginine was evident on the radiochromatograms of HUVEC cytosol and saphenous vein extracts. 3. The intracellular levels of [14C]-L-NMMA and [14C]-citrulline in SGHEC-7 cells incubated with [14C]-L-NMMA (0.5 microCi ml-1: 8.9 microM) for 1 h were 113 +/- 22 and 67.6 +/- 6.2 pmol mg-1 cell protein respectively (n = 7). Co-incubation with NGNGdimethyl-L-arginine (ADMA; 100 microM) but not NGNGdimethyl-L-arginine (SDMA; 100 microM) reduced the intracellular level of [14C]-citrulline to 26.3 +/- 3.7 pmol mg-1 cell protein (P0.01; n = 3) without reducing the intracellular level of [14C]-L-NMMA. 4. The intracellular levels of [14C]-citrulline in SGHEC-7 cells incubated with [14C]-L-NMMA for 1 h were reduced following co-incubation with NGnitro-L-arginine methylester (L-NAME; 1 mM), NGnitro-L-arginine (L-NOARG; 1 mM) and L-canavanine (1 mM) to 47.1 +/- 6.2, 24.7 +/- 3.6 and 12.5 +/- 2.8% of control levels (P0.001; n = 9). ADMA (1 mM; n = 3) reduced intracellular [14C]-citrulline levels to4 +/- 4% of control (P0.01) but SDMA (1 mM; n = 3) had no effect.5. The accumulation of endogenously synthesized ADMA in the culture supernatant of SGHEC-7 cells was increased by co-incubation with L-NMMA (1 mM) from 1.98 +/- 0.08 to 2.74 +/- 0.36 nmol mg- cell protein, an increase of 40%.6. These results demonstrate that human vasculature possesses an enzyme which has similar properties to dimethylarginase; human endothelial cells and human saphenous vein metabolize L-NMMA to citrulline via a process inhibited by ADMA but not SDMA. The increase in endothelium-derivedADMA following co-incubation with L-NMMA is consistent with competition between ADMA and L-NMMA for dimethylarginase. Inhibition of this enzyme might increase the intracellular concentration of ADMA, an endogenously produced compound that inhibits nitric oxide synthesis.

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.

Objective Maternal serum levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase, are known to be increased in pregnant women with high-resistance placental circulation in the second trimester. The aim of the present study was to investigate the relationship between uterine artery resistance and maternal serum ADMA concentrations in the first trimester. Methods Doppler ultrasound examination of the maternal uterine arteries was performed at 10–14 weeks’ gestation. High resistance was defined as bilateral uterine artery notches and a mean resistance index > 95 th centile. Control cases were defined as those presenting with no notches and a mean resistance index < 95 th centile. ADMA concentrations were measured in maternal serum. Results Forty singleton pregnancies were examined, 21 with high uterine artery resistance and 19 controls. Three cases of pre-eclampsia occurred, all in the high-resistance group. The mean (SD) maternal serum ADMA concentration was 0.78 (44%) and 0.93 (56%) µmol/L in the high-resistance and control groups, respectively (P = 0.17). There was no statistically significant correlation between maternal serum ADMA and uterine artery resistance index (ρ =− 0.15, P = 0.36). Conclusions No significant difference was found in maternal serum ADMA between pregnancies with firsttrimester high-resistance uterine artery blood flow and controls. While second-trimester circulating ADMA may have a direct link with maternal endothelial function, in the first trimester the effect of ADMA may be mainly evident at the placental level, without consequently affecting maternal circulating concentrations of the substance. Copyright  2008 ISUOG. Published by John Wiley & Sons, Ltd.

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. © The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Our understanding of important biological phenomena such as invasion, migration, and apoptosis has advanced greatly through the prudent use of in vitro models based on isolated cells in culture. Established cell lines are readily manipulated using simple molecular biological techniques and their abundance as homogenous populations allows the rapid accumulation of statistically significant data. The study of human trophoblast function in vitro has been hampered by the difficulties associated with obtaining and culturing primary human trophoblasts including the paucity of material and contamination with other cell types. This chapter describes a cheap and simple method for the production of human trophoblast cell lines using poly-L-ornithine. It details the production, isolation and initial characterization of these cells and provides simple tips on how to store and maintain a bank of cells for future needs.

Our understanding of important biological phenomena such as invasion, migration, and apoptosis has advanced greatly through the prudent use of in vitro models based on isolated cells in culture. Established cell lines are readily manipulated using simple molecular biological techniques and their abundance as homogenous populations allows the rapid accumulation of statistically significant data. The study of human trophoblast function in vitro has been hampered by the difficulties associated with obtaining and culturing primary human trophoblasts including the paucity of material and contamination with other cell types. This chapter describes a cheap and simple method for the production of human trophoblast cell lines using poly-L-ornithine. It details the production, isolation and initial characterization of these cells and provides simple tips on how to store and maintain a bank of cells for future needs.

Objective—Invasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved.Methods and Results—Spiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model.Conclusions—Extravillous trophoblast induction of endothelial apoptosis is a possible mechanism by which the endothelium is removed, and vascular remodeling may occur in uterine spiral arteries. Fas/FasL interactions have an important role in trophoblast-induced endothelial apoptosis.

Excessive apoptosis of trophoblast cells is thought to be a contributing factor in complications of pregnancy such as pre-eclampsia. Hepatocyte growth factor (HGF) inhibits apoptosis in trophoblasts and we have investigated the signalling pathways through which this anti-apoptotic effect is mediated. Treatment of cells with HGF led to rapid phosphorylation of Akt while an Akt inhibitor blocked the protective effect of HGF. Glycogen synthase kinase-3beta (GSK-3beta) was found to be one of the downstream targets of Akt. HGF treatment inactivated GSK-3beta which in turn led to the activation of the transcription factor beta-catenin. Pharmacological inhibition of GSK-3beta, independently of HGF treatment, strongly increased both beta-catenin activity and cell survival, suggesting that beta-catenin alone has a pronounced anti-apoptotic effect. We also found that both HGF treatment and pharmacological activation of beta-catenin leads to increased expression of inducible nitric oxide synthase (iNOS). We suggest that the Akt mediated activation of beta-catenin leads to inhibition of trophoblast apoptosis following increased expression of iNOS.

OBJECTIVE: Unfractionated and low-molecular-weight heparin and low-dose aspirin are used for the prevention of pregnancy loss in pregnant women with thrombophilia. We investigated the effect of these drugs on in vitro models of human extravillous trophoblast motility and differentiation. METHODS: Chorion from term placentas was digested and extravillous trophoblast isolated. Extravillous trophoblast formed giant multinuclear cells that were counted after 24, 36, and 48 hours of culture. This model was then used to investigate the effect of unfractionated, low-molecular-weight heparin and aspirin on in vitro extravillous trophoblast differentiation at both therapeutic and supratherapeutic doses. In addition, the effect of unfractionated and low-molecular-weight heparin on hepatocyte growth factor–stimulated SGHPL-4 cell (extravillous trophoblast cell line) motility was determined by time-lapse microscopy. RESULTS: At therapeutic doses unfractionated heparin promoted extravillous trophoblast differentiation. However, low-molecular-weight heparin inhibited giant multinuclear cells formation. At supratherapeutic doses, both low-molecular-weight and unfractionated heparin promoted extravillous trophoblast differentiation. Low-dose aspirin had minimal effects on the extravillous trophoblast differentiation. Both unfractionated and low-molecular-weight heparin inhibited hepatocyte growth factor–stimulated extravillous trophoblast motility at supratherapeutic doses. At a therapeutic dose of 0.25 IU/mL, only unfractionated heparin inhibited hepatocyte growth factor–stimulated motility, whereas low-molecular-weight heparin had no effect. CONCLUSION: Our data suggest that unfractionated and low-molecular-weight heparin have differing effects on trophoblast differentiation and motility at therapeutic doses. This finding may be one of many factors that contribute to the clinical scenario. LEVEL OF EVIDENCE: II-2

Background— Experimental evidence suggests that estrogens stimulate the production of nitric oxide (NO) by vascular endothelial cells. This effect has been attributed to increased expression and enzymatic activity of both the constitutive and inducible isoforms of NO synthase. In this study, we have investigated whether estrogens regulate the metabolism or release of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase. Methods and Results— The concentration of ADMA in the plasma of 15 postmenopausal women was 0.722±0.04 μmol/L (mean±SEM). Two weeks after subcutaneous implantation with estradiol, there was an increase in plasma estradiol concentration from 0.693±0.075 to 0.81±87 nmol/L, which was accompanied by a significant fall in plasma ADMA concentration to 0.588±0.03 μmol/L ( P =0.006). Human and murine endothelial cell lines previously cultured in estrogen-free medium and then exposed to 17β-estradiol showed a dose-dependent decrease in the release of ADMA. This reached statistical significance at 10 −14 mol/L 17β-estradiol and was accompanied by a corresponding increase in the activity of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catalyzes the metabolism of ADMA. Conclusions— We have demonstrated that estrogens can alter the catabolism and release of ADMA in vitro and reduce the circulating concentration in vivo. We therefore propose that increased DDAH activity and the subsequent fall in ADMA could contribute to the positive effect of estrogen on NO synthesis.

Intracellular factors that regulate nitric oxide (NO) synthesis represent important targets in tumor progression. Overexpression of dimethylarginine dimethylaminohydrolase (DDAH), which metabolizes the endogenous inhibitors of NO synthesis asymmetric dimethylarginine and N-monomethyl-L-arginine, results in C6 gliomas with enhanced growth rate compared with wild type. To investigate the effects of DDAH on tumor vascular morphogenesis in vivo, we have measured the transverse relaxation rates R(2)* and R(2) in clone D27 gliomas overexpressing DDAH and C6 wild-type gliomas using intrinsic susceptibility magnetic resonance imaging (MRI), sensitive to changes in endogenous [deoxyhemoglobin], and susceptibility contrast-enhanced MRI using the intravascular blood pool contrast agent NC100150, and we compared the results with fluorescence microscopy of the tumor uptake of the perfusion marker Hoechst 33342. The baseline R(2)* was significantly faster in the D27 tumors, consistent with a greater vascular development (P0.02, ANOVA). There was no significant difference between the response of the two tumor types to hypercapnia (5% CO(2)/95% air), used as a probe for vascular maturation, or hyperoxia (5% CO(2)/95% O(2)), used as a probe for vascular function. NC100150 increased the R(2)* and R(2) rates of both tumor types and demonstrated a significantly larger blood volume in the D27 tumors (P0.02, ANOVA). This correlated with a significantly greater uptake of Hoechst 33342 in the D27 tumors compared with C6 wild-type tumors (P0.02, ANOVA). Despite the increased tumor blood volume, the Delta R(2)*/Delta R(2) ratio, an index of microvessel size, showed that the capillaries in the two tumor types were of a similar caliber. The data highlight the potential of susceptibility MRI-derived quantitative end points to noninvasively assess tumor angiogenesis, and in this regard, the use of intravascular blood pool contrast agents such as NC100150 appears very promising. Overexpression of DDAH results in increased neovascularization of C6 gliomas in vivo. The lack of significant difference in hypercapnic/hyperoxic response between the C6 and D27 tumors and the similar vessel caliber are also consistent with a role for DDAH in the initial stages of vasculogenesis.

Apoptosis is thought to play an important regulatory role in placental development and inappropriate trophoblast apoptosis has been implicated in complications of pregnancy such as pre-eclampsia. Here we show that apoptosis of a human extravillous trophoblast-derived cell line (SGHPL-4) can be regulated by nitric oxide (NO). Nitric oxide produced exogenously by the addition of NO donors was able to delay or inhibit apoptosis induced by a combination of tumour necrosis factor alpha and actinomycin D and to suppress the activity of caspase 3. Treatment with hepatocyte growth factor (HGF) stimulated expression of the inducible isoform of NO synthase and was also able to protect SGHPL-4 cells from caspase 3 activation and apoptosis. The inhibition of basal NO production with NO synthase inhibitors was shown to sensitise cells to apoptotic stimuli and to reduce the level of endogenous caspase 3 nitrosylation. The anti-apoptotic effects of NO in these extravillous trophoblast cells appear to be mediated through the production of cyclic GMP as inhibitors of soluble guanylate cyclase inhibited the protective effect of both HGF and NO donors.

Objective— Plasma levels of an endogenous nitric oxide (NO) synthase inhibitor, asymmetric dimethylarginine (ADMA), are elevated in chronic renal failure, hypertension, and chronic heart failure. In patients with renal failure, plasma ADMA levels are an independent correlate of left ventricular ejection fraction. However, the cardiovascular effects of a systemic increase in ADMA in humans are not known. Methods and Results— In a randomized, double-blind, placebo-controlled study in 12 healthy male volunteers, we compared the effects of intravenous low-dose ADMA and placebo on heart rate, blood pressure, cardiac output, and systemic vascular resistance at rest and during exercise. We also tested the hypothesis that ADMA is metabolized in humans in vivo by dimethylarginine dimethylaminohydrolase (DDAH) enzymes. Low-dose ADMA reduced heart rate by 9.2±1.4% from 58.9±2.0 bpm ( P P P −5 ( P P P Conclusions— This study defines the cardiovascular effects of a systemic increase in ADMA in humans. These are similar to changes seen in diseases associated with ADMA accumulation. Finally, our data also indicate that ADMA is metabolized by DDAHs extensively in humans in vivo.

Background Polyamines are regulators of proliferation and differentiation in mammalian cells. They are also known to regulate cell survival and apoptosis, although their precise function varies between cell types. We have investigated the effect of polyamines on the apoptosis of human extravillous trophoblasts. Methods Using the extravillous trophoblast-derived cell line SGHPL-4 we performed time-lapse microscopy studies to evaluate the induction of apoptosis following exposure to polyamines. Results The polyamines spermine, and to a lesser extent spermidine, were able to induce apoptosis in extravillous trophoblasts. The induction of apoptosis occurred rapidly and was accompanied by DNA fragmentation and morphological changes consistent with the onset of apoptosis. Apoptosis was inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk, although no activity was detected using assays for caspase-2, -3, -6, -8 or -9 activity. We demonstrated that an oxidation product of spermine accounted for the induction of apoptosis and implicated the formation of hydrogen peroxide as this oxidation product. We have also demonstrated that exposure to nitric oxide inhibited the onset of spermine-induced apoptosis. Conclusions Spermine and spermidine induce apoptosis in extravillous trophoblasts following their oxidation and the production of hydrogen peroxide. Nitric oxide is able to inhibit this apoptosis.

Hepatocyte growth factor (HGF) increases human trophoblast motility and invasion, an effect which is abrogated when inducible nitric oxide synthase (iNOS) is inhibited. In this study we have investigated the pathways involved in the regulation of trophoblast motility. Both basal and HGF-stimulated motility of the extravillous trophoblast cell line, SGHPL-4, were inhibited in a dose-dependent manner by the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, LY294002. HGF-stimulated iNOS expression was also inhibited by LY294002 and direct activation of PI3-kinase, using the peptide 740Y-P, led to an increase in iNOS expression and cell motility. Pretreatment with rapamycin, which acts at a point distal to PI3-kinase activation, also inhibited basal and HGF-stimulated motility. Inhibition of the p42/p44 mitogen activated protein kinase (MAPK) pathway but not the p38 MAPK pathway had significant inhibitory effects on HGF-stimulated but not basal trophoblast motility. Inhibition of p42/p44 MAPK also inhibited HGF-induced iNOS expression. This data demonstrate that the PI3-kinase signaling pathway is involved in basal trophoblast motility and that both MAPK and PI3-kinase signaling pathways are important in HGF-stimulated motility and iNOS expression.

Objectives: Hepatocyte growth factor (HGF) is an angiogenic mitogen which stimulates migration in various cell types and has been shown to induce the production of nitric oxide (NO) in epithelial cells. Conflicting data exist on the effect of NO on endothelial cell migration. The aim of this study was to investigate a possible role for NO in HGF-stimulated endothelial cell motility. Methods: The study was performed primarily using an endothelial cell line derived from adult human saphenous vein. Transient transfection experiments were additionally performed using an adult human coronary artery endothelial cell line. Nitric oxide synthase expression was examined by western blot analysis. Time-lapse digital image microscopy was used to measure cell motility. A DNA construct was used in transient transfections to over-express inducible nitric oxide synthase (iNOS) as an N-terminal fusion to enhanced green fluorescent protein (EGFP). Results: HGF upregulated the expression of iNOS but not constitutive endothelial nitric oxide synthase (eNOS). Treatment of cells with the specific iNOS inhibitor 1400 W revealed that functional iNOS was required for HGF-stimulated endothelial cell motility. HGF-induced iNOS expression was partially abrogated in the presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but not the Src kinase inhibitor, PP1. Endothelial cell motility increased significantly ( P

Methylarginines inhibit nitric oxide synthases (NOS). Cellular concentrations of methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3.18). We have cloned human DDAH and identified and expressed a second novel DDAH isoform (DDAH I and II respectively). DDAH I predominates in tissues that express neuronal NOS. DDAH II predominates in tissues expressing endothelial NOS. These results strengthen the hypothesis that methylarginine concentration is actively regulated and identify molecular targets for the tissue and cell-specific regulation of methylarginine concentration.

1. Experiments were performed to examine the effects of anti-fungal imidazole compounds (clotrimazole, econazole and miconazole) on the induction of nitric oxide (NO) synthase and subsequent production of NO in the cultured murine monocyte/macrophage cell line J774 using a specific cDNA probe for inducible NO synthase mRNA and by monitoring nitrite production. 2. Stimulation of J774 cells with lipopolysaccharide (LPS, 10 micrograms ml-1) resulted in the induction of NO synthase activity as determined by nitrite accumulation in the culture medium (48 +/- 3 nmol per 10(6) cells over 24 h). Production of nitrite was inhibited by co-incubation of cells with LPS (10 micrograms ml-1) and either dexamethasone (10 microM) or NG-monomethyl-L-arginine (L-NMMA; 0.1 mM), however, only L-NMMA was an effective inhibitor of nitrite production when added after induction of NO synthase had occurred. 3. Co-incubation of J774 cells with LPS (10 micrograms ml-1) and either clotrimazole, econazole or miconazole (1-10 microM) resulted in a concentration-dependent inhibition of nitrite production over the subsequent 24 h without any evidence for a cytotoxic effect. However, addition of these imidazoles after induction of NO synthase did not inhibit nitrite production. 4. Messenger RNA for inducible NO synthase was not detected in unstimulated J774 cells. Treatment with LPS (10 micrograms ml-1) for 4 h resulted in significant expression of mRNA for inducible NO synthase which was not altered in the presence of econazole (10 microM) but was reduced significantly by dexamethasone (10 microM). 5. These results demonstrate that anti-fungal imidazoles inhibit the production of nitric oxide by cultured J774 cells by a mechanism which appears to differ from that of dexamethasone and substrate type inhibitors of NO synthase. Furthermore, the presence of mRNA for NO synthase does not indicate the presence of functionally active NO synthase.

Objective: The aim was to study whether basal or cytokine stimulated generation of nitric oxide (NO) modulates platelet adhesion to human umbilical vein endothelial cells (HUVEC). Methods: The adhesion of 111In labelled human platelets to transfected HUVEC (SGHEC-7) was measured either alone or after incubation of SGHEC-7 cells for 18 h with interleukin-1β (IL-1β) and/or tumour necrosis factor a (TNFα). The activity of NO synthase in these cells was measured by formation of citrulline. The effects of dexamethasone (0.3 μM) and NG-monomethyl-L-arginine (L-NMMA, 100 μM) on these two variables were determined. Results: Stimulation of SGHEC-7 cells with IL-1β or TNFα (each at 1-30 ng·ml−1) caused them to express the inducible NO synthase, an effect that was prevented by dexamethasone. Platelet adhesion to unstimulated SGHEC-7 cells was

The binding of 125I-labelled bovine TSH to a human thyroid cell line (SGHTL-34) has been studied. Binding to hormonally responsive cells was time dependent, specific and reversible. Scatchard analysis of the binding data indicated the presence of a single binding site with high affinity (intrinsic dissociation constant (Kd) = 0·25 ±0·08 nmol/l; mean ± s.e.m.; n = 4) and low capacity (maximum binding (Bmax) = 104±29 fmol/mg protein; mean ± s.e.m.; n = 4). Hill plots confirmed the presence of a single site. Kinetic data demonstrated close agreement between the Kd and Bmax obtained from the competition data (Kd = 0·23±0·35 nmol/l; Bmax = 161 ±83 fmol/mg protein; n = 6). Journal of Endocrinology (1990) 127, 351–356