Regulation of epithelial-mesenchymal transition by homeobox gene DLX4 in JEG-3 trophoblast cells: a role in preeclampsia

The pathogenesis of preeclampsia is unclear but is thought to be related to shallow trophoblast invasion. An invasive phenotype is acquired by trophoblasts through the process of epithelial-mesenchymal transition (EMT). We proposed that EMT in trophoblasts is deregulated in preeclampsia. The homeobox gene DLX4 plays an important role in epithelial-mesenchymal interactions during embryonic and placental development. To elucidate the role of DLX4 in trophoblast EMT and preeclampsia, we investigated the expression of DLX4 in preeclampsia-affected placentas and the effect of DLX4 on EMT in trophoblast-derived JEG-3 cells. DLX4 expression was downregulated in preeclampsia-affected placentas and hypoxic JEG-3 cells. Knockdown of DLX4 by RNA interference (RNAi) inhibited the motility and invasion ability of JEG-3 cells, decreased the expression of E-cadherin, and upregulated the expression of the E-cadherin repressor Snail. Our findings suggest that decreased expression of DLX4 leads to the pathogenesis of preeclampsia by inhibiting EMT in trophoblasts and provides new insight into the pathophysiological mechanism of preeclampsia.     

Markers of early endothelial dysfunction in intrauterine growth restriction-derived human umbilical vein endothelial cells revealed by 2D-DIGE and mass spectrometry analyses

Intrauterine growth restriction (IUGR) associates with fetal and placental vascular dysfunction, and increased cardiovascular risk later on life. We hypothesize that endothelial cells derived from IUGR umbilical veins present significant changes in the proteome which could be involved in the endothelial dysfunction associated to this conditions. To address this the proteome profile of human umbilical endothelial cells (HUVEC) isolated from control and IUGR pregnancies was compared by 2D-Differential In Gel Electrophoresis (DIGE) and further protein identification by MALDI-TOF MS. Using 2D-DIGE 124 spots were identified as differentially expressed between control and IUGR HUVEC, considering a cut-off of 2 fold change, which represented ∼10% of the total spots detected. Further identification by MALDI-TOF MS and in silico clustering of the proteins showed that those differentially expressed proteins between control and IUGR HUVEC were mainly related with cytoskeleton organization, proteasome degradation, oxidative stress response, mRNA processing, chaperones and vascular function. Finally Principal Component analysis of the identified proteins showed that differentially expressed proteins allow distinguishing between control and IUGR HUVEC based on their proteomic profile. This study demonstrates for the first time that IUGR-derived HUVEC maintained in primary culture conditions present an altered proteome profile, which could reflect an abnormal programming of endothelial function in this fetal condition.     

Deferential regulation of placenta growth factor (PlGF)-mediated signal transduction in human primary term trophoblast and endothelial cells

Increasing evidence supports that many common obstetrical complications may involve the disruption of normal placental and/or uterine vascular function. Placenta growth factor (PlGF) is an angiogenic factor that is abundantly expressed in the placenta, with primary site of synthesis being trophoblast. Receptors for PlGF include products of the fms-like tyrosine kinase (flt-1) gene which is expressed in several cell types including endothelial cells and trophoblast. PlGF activation of flt-1 in trophoblast induces the stress activated protein kinase (SAPK) signal transduction pathways, JNK (c-Jun-N-Terminal Kinase) and p38, with little induction of the extracellular signal-regulated protein kinase (ERK)-1/2 pathways. In contrast, PlGF induces strong ERK-1/2 activation, but little JNK or p38 responses in human umbilical vein endothelial cells (HUVEC). To better understand the biochemical functions of PlGF in trophoblast, we studied upstream signal regulatory molecules to determine those that are responsible for directing the divergent PlGF signal transduction responses in these cell types. PlGF induced similar activation of Nck and PLC-gamma in trophoblast and HUVEC. In marked contrast, SHP-2 and Gab2 were strongly activated by PlGF in endothelial cells but not trophoblast. These results suggest a general role for Nck and PLC-gamma in mediating PlGF signal transduction responses independent of the different downstream MAPK pathways activated. However, SHP-2 and Gab2 are regulatory molecules involved in the PlGF induction of different terminal pathways in HUVEC and trophoblast.     

Homeobox gene HLX1 is a regulator of colony stimulating factor-1 dependent trophoblast cell proliferation

The cytokine colony stimulating factor-1 (CSF-1) is a key regulator of the proliferation, differentiation and activation of mononuclear phagocytes. CSF-1 also plays an important role in reproduction. CSF-1 is produced in the placenta and activates signal transduction pathways that significantly increase the proliferation of placental trophoblast cells in culture. The target genes activated by CSF-1 mediated signal transduction in the nucleus are not well understood. Here, we use placental trophoblast cells to investigate potential downstream effector genes of CSF-1. HLX1 is a homeobox gene that controls proliferation in embryonic cell types and haematopoietic cell lineages. We have shown HLX1 is expressed in placental trophoblast cells but its functional role in the placenta is unknown. Following CSF-1 stimulation, HLX1 mRNA expression was significantly increased in SGHPL-4 and HTR-8/SVNeo cultured trophoblast cells (p<0.001, n=3). siRNA-mediated reduction of HLX1 mRNA levels with four independent oligonucleotides (siRNAs) resulted in significantly decreased cell proliferation in both cell lines (p<0.001, n=4). When HLX1 mRNA levels were reduced in the presence of CSF-1 stimulation, proliferation remained significantly decreased (p<0.001, n=4) in both the cell lines. We have shown for the first time that a homeobox gene, HLX1, is a downstream effector gene of CSF-1, that HLX1 regulates placental cell proliferation and that CSF-1 acts, at least in part, through HLX1 to control cell proliferation.     

A comparative study of the effect of three different syncytiotrophoblast micro-particles preparations on endothelial cells

Pre-eclampsia is a pregnancy-associated multi-system disorder of unknown etiology, characterized by damage to the maternal endothelium. The latter facet has been suggested to be mediated in part by elevated shedding of inflammatory placental syncytiotrophoblast micro-particles (STBM) into the maternal circulation. In this study, we have examined STBM prepared by three different methods: mechanical dissection, in vitro placental explant culture and perfusion of placental cotyledons. All three preparations yielded morphologically similar STBM, as confirmed by scanning electron microscopy, and all contained syncytiotrophoblast-specific proteins as determined by the presence of placental alkaline phosphatase. The functional properties of the three STBM preparations were examined on human umbilical vein endothelial cells (HUVEC), where the mechanically prepared particles were found to inhibit proliferation to the greatest extent. Furthermore, only mechanically prepared STBM lead to the detachment and apoptosis of HUVEC cells. Our study, therefore, suggests that STBM prepared from placental perfusion or in vitro explant culture are biologically different from mechanically prepared ones, and may provide a better approximation of physiologically produced placental micro-particles.     

Role of arginase-2 and eNOS in the differential vascular reactivity and hypoxia-induced endothelial response in umbilical arteries and veins

The main vasodilator in the placenta is nitric oxide (NO), which is synthesized by endothelial NO synthase (eNOS). Arginase-2 competes with eNOS for l-arginine, and its activity has been related with vascular dysfunction. Recently, we showed that hypoxia induces arginase-2, and decreases eNOS activity in human umbilical vein endothelial cells (HUVEC). However there is evidence that vascular responses to hypoxia are not similar throughout the placental vascular tree. We studied whether arginase-2 plays a role controlling vascular tone in human umbilical vessels, and the changes in the expression of arginase-2 and eNOS proteins by hypoxia in endothelial cells from umbilical arteries (HUAEC) and veins (HUVEC). In isolated umbilical vessels the presence of eNOS and arginase-2 was determined in the endothelium, and the NO-dependent vasoactive responses in the presence and absence of S-(2-boronoethyl)-L-cysteine (BEC, arginase inhibitor) were studied. Additionally, HUAEC and HUVEC were exposed (0-24 h) to hypoxia (2% O2) or normoxia (5% O2), and protein levels of eNOS (total and phosphorylated at serine-1177) and arginase-2 were determined. In umbilical arteries and veins arginase-2 and eNOS were detected mainly at the endothelium. BEC induced a higher concentration-dependent relaxation in umbilical arteries than veins, and these responses were NOS-dependent. In HUAEC exposed to hypoxia there were no changes in eNOS and arginase-2 levels, however there was a significant increase of p-eNOS. In contrast, HUVEC showed an increase in arginase-2 and a reduction of p-eNOS in response to hypoxia. These results show that arginases have a vascular role in placental vessels counteracting the NOS-dependent relaxation, which is differentially regulated in placental artery and vein endothelial cells.     

Nicotine and cotinine affect the release of vasoactive factors by trophoblast cells and human umbilical vein endothelial cells

Objective

To examine nicotine (N) and cotinine (C) effects on trophoblast cells (TCs) and human umbilical vein endothelial cells (HUVEC) secretion of soluble fms-like tyrosine kinase (sFlt-1), soluble endoglin (sENG), placental growth factor (PlGF), transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF).

Study design

Human placentas and umbilical cords were collected from uncomplicated pregnancies at term from a total of 24 non-smoking women with a history of normal blood pressure. TCs and HUVEC were cultured for 24 h with C or N (from 10⁻¹² to 10⁻⁷ M).

Main outcome measures

sFlt-1, sENG, PlGF, TGF-beta and VEGF release and messenger RNA (mRNA) expression were evaluated by ELISA and real-time polymerase chain reaction (PCR), respectively.

Results

N and C reduced sFlt-1, sENG and PlGF release by TCs and TGF-beta release by HUVEC. Conversely, N and C increased PlGF secretion, while N alone increased sFlt-1 release by HUVEC. N and C were able to modulate VEGF mRNA expression in HUVEC.

Conclusions

Our results suggest that N and C affect the balance of some important vasoactive factors released by TCs and HUVEC. This might be one of the possible mechanism through which smoke reduces the risk of hypertensive disorders during pregnancy as well as contributes to the well known detrimental effects of smoking on fetal development.     

Stimulation of DNA Synthesis in Trophoblasts and Human Umbilical Vein Endothelial Cells by Hepatocyte Growth Factor Bound to Extracellular Matrix

Hepatocyte growth factor (HGF) promotes the growth not only of hepatocytes but also of several other types of cells such as cytotrophoblasts and endothelial cells. Recent studies have revealed that HGF is trapped in the extracellular (ECM) matrix through heparan sulphate in vivo, thereby acting as a mitogen for hepatocytes in cooperation with heparan sulphate. In this study, we detected HGF protein in chorionic tissue and placental tissue extracts, and found that HGF and heparan sulphate were co-distributed in the endothelial basement membrane and trophoblast basement membrane on immunohistochemical examination. The rates of DNA synthesis in primary cultured cytotrophoblasts and human umbilical vein endothelial cells (HUVEC) cultured on HGF-bound Matrigeltrade mark were 6-8 times those of control cytotrophoblasts and HUVEC. When Matrigeltrade mark dishes were pretreated with heparinase and heparitinase prior to binding of HGF, stimulation of DNA synthesis was markedly decreased. A considerable decrease in stimulation of DNA synthesis was observed following washing of HGF-bound Matrigeltrade mark with 1 m acetic acid, 1 m NaCl and 0.1 per cent trypsin, but not following treatment with chondroitinase ABC. These observations suggest that HGF can be trapped in ECM in vivo, thereby acting as a mitogen for cytotrophoblasts and placental vein endothelial cells in cooperation with heparan sulphate.     

Expression of homeobox gene transcripts in trophoblastic cells

OBJECTIVE: This study was conducted to examine the dynamics of homeobox gene expression in the differentiation of trophoblasts as a key to the understanding of the regulatory mechanisms that are involved in placental development. STUDY DESIGN: Expression of homeobox genes was examined in primary trophoblastic cells and in the BeWo choriocarcinoma model cell lines by molecular and immunocytochemistry techniques. RESULTS: We demonstrated the expression of 3 homeobox genes (HOX B6, HOX C6, and HOX A11) in primary trophoblastic cells. BeWo cells showed an expression pattern similar to that of the primary cell lines. In both primary trophoblasts and BeWo cells, the HOX A11 gene, but not the HOX B6 or HOX C6 genes, were found to down-regulate with differentiation from single- to multinucleate giant cells. CONCLUSION: This study demonstrates a novel expression pattern for HOX A11 gene in trophoblastic differentiation and suggests that the down-regulation of HOX A11 may be necessary for the differentiation of cytotrophoblasts into syncytiotrophoblasts.     

Reduced l-Arginine Transport and Nitric Oxide Synthesis in Human Umbilical Vein Endothelial Cells from Intrauterine Growth Restriction Pregnancies is Not Further Altered by Hypoxia

Intrauterine growth restriction (IUGR) is associated with chronic fetal hypoxia, altered placental vasodilatation and reduced endothelial nitric oxide synthase (eNOS) activity. In human umbilical vein endothelial cells (HUVEC) from pregnancies complicated with IUGR (IUGR cells) and in HUVEC from normal pregnancies (normal cells) cultured under hypoxia l-arginine transport is reduced; however, the mechanisms leading to this dysfunction are unknown. We studied hypoxia effect on l-arginine transport and human cationic amino acid transporters 1 (hCAT-1) expression, and the potential NO and protein kinase C α (PKCα) involvement. Normal or IUGR HUVEC monolayers were exposed (0–24 h) to 5% O₂ (normoxia), and 1 or 2% O₂ (hypoxia). l-Arginine transport and hCAT-1 expression, phosphorylated and total PKCα or eNOS protein and mRNA expression were quantified. eNOS involvement was tested using a siRNA against eNOS (eNOS-siRNA) adenovirus. IUGR cells in normoxia or hypoxia, and normal cells in hypoxia exhibited reduced l-arginine transport, hCAT-1 expression, NO synthesis and eNOS phosphorylation at Serine¹¹⁷⁷, effects reversed by calphostin C (PKC inhibitor) and S-nitroso-N-acetyl-l,d-penicillamine (SNAP, NO donor). However, N^G-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor) reduced hCAT-1 expression only in normal cells in normoxia. Increased Thr⁶³⁸-phosphorylated PKCα was exhibited by IUGR cells in normoxia or hypoxia and normal cells in hypoxia. The effects of hypoxia in normal cells were mimicked in eNOS-siRNA transduced cells; however, IUGR phenotype was unaltered by eNOS knockdown. Thus, IUGR- and hypoxia-reduced l-arginine transport could result from increased PKCα, but reduced eNOS activity leading to a lower hCAT-1 expression in HUVEC. In addition, IUGR endothelial cells are either not responsive or maximally affected by hypoxia. These mechanisms could be responsible for placental dysfunction in diseases where fetal endothelium is chronically exposed to hypoxia, such as IUGR.