Decidual natural killer cells regulate vessel stability: implications for impaired spiral artery remodelling.

Decidual NK (dNK) cells are present during uterine spiral artery remodelling, an event that is crucial for successful placentation and the provision of an adequate blood supply to the developing fetus. Spiral artery remodelling is impaired in the pregnancy complication pre-eclampsia. Although dNK cells are known to play active roles at the maternal-fetal interface, little is known about their effect on endothelial integrity, an important component of vessel stability. We present a study in which we have modelled dNK-endothelium interactions, using first-trimester dNK cells isolated from both normal pregnancies and those with impaired spiral artery remodelling. dNK cells were isolated from first-trimester pregnancies, screened by uterine artery Doppler ultrasound to determine resistance indices (RI) that relate to the extent of spiral artery remodelling. dNK culture supernatant from normal-RI pregnancies (but not high-RI pregnancies) destabilised endothelial tube-like structures in Matrigel, and normal-RI dNK cells induced endothelial intercellular adhesion molecule-1 and tumour necrosis factor-α expression to a greater extent than high-RI dNK cells. We have established a functional role for dNK cells in the disruption of endothelial structures and have suggested how impairment of this process may be contributing to the reduced vessel remodelling in pregnancies with a high uterine artery resistance index. These findings have implications for our understanding of the pathology of pre-eclampsia and other pregnancy disorders where remodelling is impaired.

Active site mutant dimethylarginine dimethylaminohydrolase 1 expression confers an intermediate tumour phenotype in C6 gliomas.

Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes the endogenous inhibitor of nitric oxide synthesis, asymmetric dimethylarginine (ADMA). Constitutive over-expression of DDAH1, the isoform primarily associated with neuronal nitric oxide synthase (nNOS) results in increased tumour growth and vascularization, and elevated VEGF secretion. To address whether DDAH1-mediated tumour growth is reliant upon the enzymatic activity of DDAH1, cell lines expressing an active site mutant of DDAH1 incapable of metabolizing ADMA were created. Xenografts derived from these cell lines grew significantly faster than those derived from control cells, yet not as fast as those over-expressing wild-type DDAH1. VEGF expression in DDAH1 mutant-expressing tumours did not differ from control tumours but was significantly lower than that of wild-type DDAH1-over-expressing tumours. Fluorescence microscopy for CD31 and pimonidazole adduct formation demonstrated that DDAH1 mutant-expressing tumours had a lower endothelial content and demonstrated less hypoxia, respectively, than wild-type DDAH1-expressing tumours. However, there was no difference in uptake of the perfusion marker Hoechst 33342. Non-invasive multiparametric quantitative MRI, including the measurement of native T(1) and T(2) relaxation times and apparent water diffusion coefficient, was indicative of higher cellularity in DDAH1-expressing xenografts, which was confirmed by histological quantification of necrosis. C6 xenografts expressing active site mutant DDAH1 displayed an intermediate phenotype between tumours over-expressing wild-type DDAH1 and control tumours. These data suggest that enhanced VEGF expression downstream of DDAH1 was dependent upon ADMA metabolism, but that the DDAH1-mediated increase in tumour growth was only partially dependent upon its enzymatic activity, and therefore must involve an as-yet unidentified mechanism. DDAH1 is an important mediator of tumour progression, but appears to have addition roles independent of its metabolism of ADMA, which need to be considered in therapeutic strategies targeted against the NO/DDAH pathway in cancer.

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.

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.

Epidermal growth factor-stimulated extravillous cytotrophoblast motility is mediated by the activation of PI3-K, Akt and both p38 and p42/44 mitogen-activated protein kinases.

BACKGROUND: Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy. METHODS AND RESULTS: In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3beta as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3beta. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3beta phosphorylation and the subsequent expression of beta-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype. CONCLUSION: We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of beta-catenin alone was insufficient to stimulate cell motility.

S-nitrosylation of proteins at the leading edge of migrating trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion.

Nitric oxide regulates many important cellular processes including motility and invasion. Many of its effects are mediated through the modification of specific cysteine residues in target proteins, a process called S-nitrosylation. Here we show that S-nitrosylation of proteins occurs at the leading edge of migrating trophoblasts and can be attributed to the specific enrichment of inducible nitric oxide synthase (iNOS/NOS2) in this region. Localisation of iNOS to the leading edge is co-incidental with a site of extensive actin polymerisation and is only observed in actively migrating cells. In contrast endothelial nitric oxide synthase (eNOS/NOS3) shows distribution that is distinct and non-colocalised with iNOS, suggesting that the protein S-nitrosylation observed at the leading edge is caused only by iNOS and not eNOS. We have identified MMP-9 as a potential target for S-nitrosylation in these cells and demonstrate that it co-localises with iNOS at the leading edge of migrating cells. We further demonstrate that iNOS plays an important role in promoting trophoblast invasion, which is an essential process in the establishment of a successful pregnancy.

Increased apoptosis in first trimester extravillous trophoblasts from pregnancies at higher risk of developing preeclampsia.

Preeclampsia complicates 5 to 10% of pregnancies and is a leading cause of maternal and fetal mortality and morbidity. Although the cause is unknown, inadequate invasion and remodeling of maternal uterine arteries by extravillous trophoblasts (EVTs) in the first trimester is a common feature. Uterine spiral artery resistance as detected by Doppler ultrasound is commonly used in the second trimester to identify pregnancies destined to develop preeclampsia. Correlation between high uterine resistance and the failure of trophoblast invasion has been reported as early as 12 weeks. However, the reason for this failure has not been established. Understanding the processes involved would significantly improve our diagnostic potential. In this study, we correlated increased first trimester uterine artery resistance with a biological abnormality in trophoblast function. EVTs derived from high-resistance pregnancies were more sensitive to apoptotic stimuli than those from normal-resistance pregnancies. Survival of EVTs from high-resistance pregnancies could be increased by nitric oxide, whereas inhibition of nitric oxide in cells from normal-resistance pregnancies increased apoptotic sensitivity. This predates the onset of symptoms by several weeks and provides evidence for a mechanism responsible for the incomplete uterine vessel remodeling and the differences in artery resistance between preeclamptic and normal pregnancies.

Invasive trophoblasts stimulate vascular smooth muscle cell apoptosis by a fas ligand-dependent mechanism.

During pregnancy, trophoblasts migrate from the placenta into uterine spiral arteries, transforming them into wide channels that lack vasoconstrictive properties. In pathological pregnancies, this process is incomplete. To define the fundamental events involved in spiral artery remodeling, we have studied the effect of trophoblasts on vascular smooth muscle cells (SMCs). Here we demonstrate for the first time that apoptosis of SMCs can be initiated by invading trophoblasts. When trophoblasts isolated from normal placenta (primary trophoblasts) or conditioned medium was perfused into spiral or umbilical artery segments, apoptosis of SMCs resulted. Culture of human aortic SMCs (HASMCs) with primary trophoblasts, primary trophoblast-conditioned medium, or a trophoblast-derived cell line (SGHPL-4) also significantly increased SMC apoptosis. Fas is expressed by spiral artery SMCs, and a Fas-activating antibody triggered HASMC apoptosis. Furthermore, a Fas ligand (FasL)-blocking antibody significantly inhibited HASMC apoptosis induced by primary trophoblasts, SGHPL-4, or trophoblast-conditioned medium. Depleting primary trophoblast-conditioned medium of FasL also abrogated SMC apoptosis in vessels in situ. These results suggest that apoptosis triggered by the release of soluble FasL from invading trophoblasts contributes to the loss of smooth muscle from the walls of spiral arteries during pregnancy.

Production of human trophoblast cell lines.

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.

Trophoblast apoptosis is inhibited by hepatocyte growth factor through the Akt and beta-catenin mediated up-regulation of inducible nitric oxide synthase.

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.

Uterine spiral artery remodeling involves endothelial apoptosis induced by extravillous trophoblasts through Fas/FasL interactions.

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.

Effects of low-molecular-weight and unfractionated heparin on trophoblast function.

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.

Overexpression of dimethylarginine dimethylaminohydrolase enhances tumor hypoxia: an insight into the relationship of hypoxia and angiogenesis in vivo.

The oxygenation status of tumors derived from wild-type C6 glioma cells and clone D27 cells overexpressing dimethylarginine dimethylaminohydrolase (DDAH) was assessed in vivo using a variety of direct and indirect assays of hypoxia. Clone D27 tumors exhibit a more aggressive and better-vascularized phenotype compared to wild-type C6 gliomas. Immunohistochemical analyses using the 2-nitroimidazole hypoxia marker pimonidazole, fiber optic OxyLite measurements of tumor pO2, and localized 31P magnetic resonance spectroscopy measurements of tumor bioenergetic status and pH clearly demonstrated that the D27 tumors were more hypoxic compared to C6 wild type. In the tumor extracts, only glucose concentrations were significantly lower in the D27 tumors. Elevated Glut-1 expression, a reliable functional marker for hypoxia-inducible factor-1-mediated metabolic adaptation, was observed in the D27 tumors. Together, the data show that overexpression of DDAH results in C6 gliomas that are more hypoxic compared to wild-type tumors, and point strongly to an inverse relationship of tumor oxygenation and angiogenesis in vivo--a concept now being supported by the enhanced understanding of oxygen sensing at the molecular level.

Estrogen stimulates dimethylarginine dimethylaminohydrolase activity and the metabolism of asymmetric dimethylarginine.

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 micromol/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 micromol/L (P=0.006). Human and murine endothelial cell lines previously cultured in estrogen-free medium and then exposed to 17beta-estradiol showed a dose-dependent decrease in the release of ADMA. This reached statistical significance at 10-14 mol/L 17beta-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.

Effects of overexpression of dimethylarginine dimethylaminohydrolase on tumor angiogenesis assessed by susceptibility magnetic resonance imaging.

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 (P < 0.02, 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 (P < 0.02, ANOVA). This correlated with a significantly greater uptake of Hoechst 33342 in the D27 tumors compared with C6 wild-type tumors (P < 0.02, 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.

Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation.

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.

Nitric oxide inhibits polyamine-induced apoptosis in the human extravillous trophoblast cell line SGHPL-4.

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-induced endothelial cell motility is mediated by the upregulation of inducible nitric oxide synthase 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<0.0001) in the presence of the exogenous NO donor spermine-NO and cells expressing the iNOS-EGFP fusion protein exhibited significantly greater (P=0.0038) motility than those expressing EGFP alone. CONCLUSIONS: These combined data show that elevated NO production is sufficient to stimulate endothelial cell motility and link HGF and NO, both previously implicated in modulating motility, in a common signalling pathway.

all-trans-Retinoic acid increases nitric oxide synthesis by endothelial cells: a role for the induction of dimethylarginine dimethylaminohydrolase.

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.