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1.
Placenta ; 32(9): 687-693, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21767875

ABSTRACT

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.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 2/metabolism , Glucocorticoids/metabolism , Hydrocortisone/metabolism , Placenta/metabolism , Pre-Eclampsia/metabolism , Cortisone/metabolism , Female , Humans , Pre-Eclampsia/etiology , Pregnancy , Pregnancy Trimester, First , Risk , Ultrasonography, Prenatal
2.
Placenta ; 31(6): 465-74, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20359743

ABSTRACT

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.


Subject(s)
Arteries/metabolism , Placenta/blood supply , Trophoblasts/metabolism , Uterus/blood supply , Apoptosis , Extracellular Matrix/metabolism , Female , Humans , Placenta/metabolism , Pregnancy
3.
Hum Reprod ; 22(9): 2523-7, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17611187

ABSTRACT

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.


Subject(s)
Heparin, Low-Molecular-Weight/pharmacology , Heparin/pharmacology , Trophoblasts/drug effects , Cell Line , Cell Movement/drug effects , Female , Humans , Pregnancy , Pregnancy Trimester, First , Trophoblasts/physiology
4.
Hum Reprod ; 21(10): 2530-7, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16920729

ABSTRACT

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.


Subject(s)
Amidohydrolases/genetics , Amidohydrolases/metabolism , Nitric Oxide/physiology , Placenta/physiology , Trophoblasts/physiology , Base Sequence , DNA Primers , Female , Humans , Keratin-7 , Keratins/analysis , Organ Culture Techniques , Placenta/cytology , Placenta/enzymology , Polymerase Chain Reaction , Pregnancy , Pregnancy Trimester, First
5.
Placenta ; 27(2-3): 137-47, 2006.
Article in English | MEDLINE | ID: mdl-15921739

ABSTRACT

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection in the United States and intrauterine transmission of HCMV occurs in approximately 40% of pregnant women with primary HCMV infection. Although HCMV infection during pregnancy clearly may be detrimental to fetal development, its consequences on placentation remain largely unexplored. In this study, the effects of HCMV infection on cytotrophoblast (CTB) invasion were investigated utilizing the first trimester extravillous CTB cell line SGHPL-4. HCMV infection significantly inhibited SGHPL-4 proliferation, epidermal growth factor (EGF)- and hepatocyte growth factor (HGF)-induced migration and invasion, as well as the secretion of matrix metalloproteinase (MMP)-2 and MMP-9. Both HCMV and EGF activated the EGF receptor (EGFR), inducing receptor tyrosine phosphorylation at specific residues. Of interest, EGFR was differentially activated by HCMV, and viral gene transcription was not required for the observed inhibitory effect on CTB invasiveness. These findings demonstrate that HCMV infection impairs CTB differentiation along the invasive pathway and that the differential regulation of EGFR by HCMV may contribute to impaired CTB function. Elucidating the mechanisms by which HCMV impairs placentation may be key in understanding fetal and maternal pathologies associated with intrauterine HCMV infection.


Subject(s)
Cell Movement , Cytomegalovirus Infections/metabolism , Cytomegalovirus/physiology , Placenta/virology , Trophoblasts/virology , Cell Line , Cell Proliferation , Cytomegalovirus/genetics , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Viral , Genes, Viral , Humans , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Phosphorylation , Placenta/cytology , Placenta/metabolism , Pregnancy , Pregnancy Trimester, First , Transcriptional Activation , Trophoblasts/cytology , Trophoblasts/physiology
6.
Placenta ; 26(10): 709-20, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16226120

ABSTRACT

Human trophoblast research relies on a combination of in vitro models, including isolated primary cultures, explant cultures, and trophoblast cell lines. In the present study, we have utilized the rotating wall vessel (RWV) bioreactor to generate a three-dimensional (3-D) model of human placentation for the study of cytotrophoblast (CTB) invasion. The RWV supported the growth of the human CTB cell line SGHPL-4 and allowed for the formation of complex, multilayered 3-D aggregates that were morphologically, phenotypically, and functionally distinct from SGHPL-4 monolayers. The cells cultured three-dimensionally differentiated into an aggressively invasive cell population characterized by the upregulation of matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-9 and urokinase-type plasminogen activator (uPA) secretion and activation. Microarray analysis of the 3-D and 2-D cultured cells revealed increased expression in the 3-D cells of various genes that are known mediators of invasion, including MT1-MMP, PECAM-1 and L-selectin, as well as genes not previously associated with CTB differentiation such as MMP-13 and MT5-MMP. These results were verified by quantitative real-time PCR. These findings suggest that when cultured in 3-D, SGHPL-4 cells closely mimic differentiating in utero CTBs, providing a novel approach for the in vitro study of the molecular mechanisms that regulate CTB differentiation and invasion.


Subject(s)
Placentation/physiology , Trophoblasts/cytology , Bioreactors , Blotting, Western , Cell Aggregation/physiology , Cell Differentiation/physiology , Cell Growth Processes/physiology , Cell Line , Female , Humans , L-Selectin/biosynthesis , L-Selectin/genetics , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/metabolism , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Platelet Endothelial Cell Adhesion Molecule-1/biosynthesis , Platelet Endothelial Cell Adhesion Molecule-1/genetics , Pregnancy , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Trophoblasts/enzymology , Trophoblasts/metabolism , Trophoblasts/ultrastructure , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolism
7.
Placenta ; 23(10): 699-705, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12398809

ABSTRACT

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.


Subject(s)
Hepatocyte Growth Factor/pharmacology , Transforming Growth Factor beta/pharmacology , Trophoblasts/physiology , Blotting, Western , Cell Division , Cell Line , Cell Movement , Culture Media , Female , Fetal Blood , Gene Expression , Humans , Luciferases/genetics , Nitric Oxide Synthase/analysis , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Pregnancy , Promoter Regions, Genetic , Recombinant Fusion Proteins , Transfection , Transforming Growth Factor beta1 , Trophoblasts/cytology
8.
Br J Cancer ; 87(6): 673-80, 2002 Sep 09.
Article in English | MEDLINE | ID: mdl-12237779

ABSTRACT

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.


Subject(s)
Amidohydrolases/metabolism , Brain Neoplasms/blood supply , Brain Neoplasms/pathology , Endothelial Growth Factors/metabolism , Endothelium, Vascular/metabolism , Glioma/blood supply , Glioma/pathology , Lymphokines/metabolism , Neovascularization, Pathologic , Nitric Oxide/metabolism , Amidohydrolases/genetics , Amidohydrolases/pharmacology , Animals , Astrocytoma/metabolism , Astrocytoma/pathology , Blotting, Northern , Blotting, Western , Brain Neoplasms/metabolism , Cell Division/physiology , Cell Movement , Cells, Cultured , Cyclic GMP/metabolism , DNA Primers/chemistry , Enzyme-Linked Immunosorbent Assay , Female , Glioblastoma/metabolism , Glioblastoma/pathology , Glioma/metabolism , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Neovascularization, Pathologic/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
10.
Placenta ; 23(2-3): 232-5, 2002.
Article in English | MEDLINE | ID: mdl-11945091

ABSTRACT

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.


Subject(s)
Chorionic Villi/blood supply , Models, Biological , Trophoblasts/physiology , Adult , Arteries/physiology , Cell Communication/physiology , Cell Line , Female , Gestational Age , Humans , Pregnancy
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