Amy Chui

The Expression of Placental Proteoglycans in Pre-Eclampsia

Background/Aims: Pre-eclampsia (PE) is one of the leading causes of maternal and perinatal morbidity and mortality. PE is defined clinically as the onset of maternal hypertension and proteinuria following 20 weeks of gestation. It is associated with altered maternal uterine decidual spiral artery remodelling, which may lead to reduced blood flow and increased thrombosis within the uteroplacental vasculature. Proteoglycans (PGs) are macromolecules which have (in combination with glycosaminoglycans) important anticoagulant roles in vascular endothelial environments, including the uteroplacental circulation. The hypothesis under consideration in this study was that differential expression of placental PGs may be associated with PE. Methods: PE and control placental samples were collected with ethics approval and patient consent. RNA and protein were extracted and real-time PCR and Western immunoblotting were performed to determine the expression of the PGs in the samples. Results: Of the nine PGs investigated, none showed increased expression, whereas the mRNA and protein expression of five of them was significantly decreased in the placentae of pre-eclamptic women compared to gestation-matched controls. Conclusion: Therefore, the results of this study support the hypothesis that a placental PG deficiency may contribute to the placental thrombotic lesions characteristic of PE.

Homeobox gene distal-less 3 is expressed in proliferating and differentiating cells of the human placenta

DLX3, a member of the large homeobox gene family of transcription factors, is necessary for normal placentation. Targeted deletion of dlx3 in mouse resulted in embryonic death due to placental failure. This study demonstrates the presence of DLX3 mRNA expression in human first trimester and term placental tissue, cultured trophoblast-like cell lines and in isolated primary villous and extravillous trophoblast cells. Using an ovine polyclonal antibody, the spatial distribution was identified for DLX3 in human placental tissues, trophoblast cell lines and in freshly isolated primary trophoblast cells. A 50 kDa immunoreactive DLX3 protein was detected in the human placenta, in trophoblast cell lines and in primary trophoblast cells. Nuclear expression for DLX3 was observed in villous cytotrophoblasts, syncytiotrophoblast and extravillous cytotrophoblast in the proximal regions of the cytotrophoblast cell columns in first trimester placental tissues. Immunoreactivity was also detected in few stromal cells and microvascular endothelial cells surrounding the fetal capillaries. In the first trimester placental bed, DLX3 expression was predominantly observed in the cytoplasm of the endovascular and interstitial trophoblasts. We conclude that the cellular expression of DLX3 was extensive in the human placenta and propose that DLX3 may play an important role in normal placental development.

Homeobox genes and down-stream transcription factor PPARγ in normal and pathological human placental development

The placenta provides critical transport functions between the maternal and fetal circulations during intrauterine development. Formation of this interface is controlled by nuclear transcription factors including homeobox genes. Here we summarize current knowledge regarding the expression and function of homeobox genes in the placenta. We also describe the identification of target transcription factors including PPARγ, biological pathways regulated by homeobox genes and their role in placental development. The role of the nuclear receptor PPARγ, ligands and target genes in human placental development is also discussed. A better understanding of these pathways will improve our knowledge of placental cell biology and has the potential to reveal new molecular targets for the early detection and diagnosis of pregnancy complications including human fetal growth restriction.

Homeobox gene Distal-Less 3 is a regulator of villous cytotrophoblast differentiation and its expression is increased in human idiopathic foetal growth restriction

Human idiopathic foetal growth restriction (FGR) is frequently associated with placental insufficiency. In our previous studies, we have reported the isolation and characterisation of the homeobox gene Distal-less 3 (DLX3) in the human placenta. In this study, we have investigated the level of DLX3 expression in idiopathic FGR-affected placentae and determined its functional role in villous trophoblast differentiation. FGR-affected placentae (n = 25) were collected based on well-defined clinical criteria and matched for gestation with control uncomplicated pregnancies (n = 25). Real-time polymerase chain reaction and immunoblotting showed increased DLX3 mRNA and protein expression in FGR-affected placentae compared with gestation-matched controls. Qualitative immunohistochemistry revealed DLX3 localisation in the syncytiotrophoblast, cytotrophoblasts and endothelial cells surrounding the foetal capillaries in both FGR-affected and control placentae. Down-regulation of DLX3 in primary villous trophoblast cells and a trophoblast-derived cell line showed decreased expression of differentiation markers, 3βHSD, βhCG and syncytin. Therefore, we conclude that increased DLX3 expression in FGR may contribute to trophoblast dysfunction observed in FGR.

Placental Syndecan Expression Is Altered in Human Idiopathic Fetal Growth Restriction

Pregnancy represents a hypercoagulable state characterized by increased thrombin generation. However, placentas from fetal growth restriction (FGR) pregnancies are characterized by increased fibrin deposition and thrombi in the vasculature, indicative of a further increase in thrombin activation and a disturbance in coagulation in this clinical setting. The cause of the coagulation disturbance observed in FGR pregnancies is currently unknown. Anticoagulant mechanisms are crucial in the regulation of thrombin activity, and current evidence suggests that syndecans are the principal placental anticoagulant proteoglycans. The aim of this study was to determine the localization, distribution, and expression of syndecans 1 to 4 in placentas complicated by idiopathic FGR compared with gestation-matched controls. Immunohistochemistry results revealed that all of the syndecans were localized to cells located closely to the maternal and fetal circulation. The mRNA and protein expression levels of both syndecan 1 and syndecan 2 were significantly decreased in FGR samples compared with controls. This is the first study to demonstrate the differential expression of syndecans 1 to 4 in idiopathic FGR placentas compared with controls. Reduced levels of syndecan expression may result in increased placental thrombosis in the uteroplacental circulation and may therefore contribute to the pathogenesis of FGR.

Altered decorin leads to disrupted endothelial cell function: A possible mechanism in the pathogenesis of fetal growth restriction?

OBJECTIVE Fetal growth restriction (FGR) is a key cause of adverse pregnancy outcome where maternal and fetal factors are identified as contributing to this condition. Idiopathic FGR is associated with altered vascular endothelial cell functions. Decorin (DCN) has important roles in the regulation of endothelial cell functions in vascular environments. DCN expression is reduced in FGR. The objectives were to determine the functional consequences of reduced DCN in a human microvascular endothelial cell line model (HMVEC), and to determine downstream targets of DCN and their expression in primary placental microvascular endothelial cells (PLECs) from control and FGR-affected placentae. APPROACH Short-interference RNA was used to reduce DCN expression in HMVECs and the effect on proliferation, angiogenesis and thrombin generation was determined. A Growth Factor PCR Array was used to identify downstream targets of DCN. The expression of target genes in control and FGR PLECs was performed. RESULTS DCN reduction decreased proliferation and angiogenesis but increased thrombin generation with no effect on apoptosis. The array identified three targets of DCN: FGF17, IL18 and MSTN. Validation of target genes confirmed decreased expression of VEGFA, MMP9, EGFR1, IGFR1 and PLGF in HMVECs and PLECs from control and FGR pregnancies. CONCLUSIONS Reduction of DCN in vascular endothelial cells leads to disrupted cell functions. The targets of DCN include genes that play important roles in angiogenesis and cellular growth. Therefore, differential expression of these may contribute to the pathogenesis of FGR and disease states in other microvascular circulations.

The Role of Placental Glycosaminoglycans in the Prevention of Pre-Eclampsia

Uncomplicated pregnancies represent a hypercoagulable state. However, placental thrombosis is rare in these pregnancies which suggest that thrombin generation must be tightly regulated. In contrast, pregnancy disorders such as pre-eclampsia (PE) demonstrate an exaggerated increase in pro-coagulant activity contributing to the thrombotic lesions observed in the uteroplacental circulation of these pregnancies. Therefore, the tight haemostatic regulation observed in uncomplicated pregnancies is disrupted in PE-affected pregnancies. Glycosaminoglycans (GAGs) are abundantly expressed within the placenta. GAGs do not exist in isolation, but are predominately bound to proteins known as proteoglycans (PGs). PGs have important anticoagulant, antiinflammatory and pro-angiogenic properties. Placentae from pregnancies complicated by PE demonstrate a reduction in PG expression. This may be a plausible explanation for the increase in thrombosis observed in this pregnancy complication. Heparin is a well known pharmacological GAG with biochemical structure similar to the endogenous GAG, heparan sulphate (HS). Recent clinical studies have suggested that antenatal heparin therapy may reduce the likelihood of developing PE and fetal growth restriction (FGR). However, the mechanism by which heparin acts to reduce the likelihood of such pregnancy complications is very poorly understood and is necessary in order to improve the efficacy of this drug before it is to be used as a standard treatment.

Downstream targets of the homeobox gene DLX3 are differentially expressed in the placentae of pregnancies affected by human idiopathic fetal growth restriction.

Human idiopathic fetal growth restriction (FGR) is associated with placental insufficiency. Previously, we reported that the expression of homeobox gene Distal-less 3 (DLX3) is increased in idiopathic FGR placentae and is a regulator of villous trophoblast differentiation. Here, we identify the downstream targets of DLX3 in trophoblast-derived cell lines. We modelled the high levels of DLX3 in FGR using an over-expression plasmid construct and complemented this using short-interference RNA (siRNA) for inactivation in cultured cells. Using a real-time PCR-based gene profiling, candidate target genes of DLX3 over-expression and inactivation were identified as regulators of trophoblast differentiation; GATA2 and PPARγ. The expression of GATA2 and PPARγ were further assessed in placental tissues and showed increased mRNA and protein levels in FGR-affected tissues compared with gestation-matched controls. We conclude that DLX3 orchestrates the expression of multiple regulators of trophoblast differentiation and that expression of these regulatory genes is abnormal in FGR.

Antiangiogenic effects of decorin restored by unfractionated, low molecular weight, and nonanticoagulant heparins

Pregnancies affected by preeclampsia (PE) or fetal growth restriction (FGR) display increases in thrombin generation and reductions in angiogenesis and cell growth. There is significant interest in the potential for low molecular weight heparins (LMWHs) to reduce the recurrence of PE and FGR. However, LMWH is associated with an increased risk of bleeding. Therefore, it is of vital importance to determine the exact molecular function of heparins in pregnancy if they are used as therapy for pregnant women. We aimed to determine this using our model for PE/FGR in microvascular endothelial cells. The expression of decorin, a proteoglycan, was reduced to mimic PE/FGR in these cells compared with controls. Four concentrations of unfractionated heparin (UFH), LMWH, and nonanticoagulant heparin (NAC) were added to determine the effect on thrombin generation, angiogenesis, and cell growth. Treatment with UFH and LMWH reduced thrombin generation and restored angiogenesis but decreased cell growth. Treatment with NAC did not affect thrombin generation, restored angiogenesis, and showed a trend toward cell growth. In conclusion, treatment with NAC produced the same, if not better, results as treatment with UFH or LMWH, without the same impact on coagulation. Therefore, NAC could potentially be a better therapeutic option for prevention of PE/FGR in high-risk women, without the risk of the adverse effects of traditional anticoagulants.

Expression of Biglycan in First Trimester Chorionic Villous Sampling Placental Samples and Altered Function in Telomerase-Immortalized Microvascular Endothelial Cells

Objective— Biglycan (BGN) has reduced expression in placentae from pregnancies complicated by fetal growth restriction (FGR). We used first trimester placental samples from pregnancies with later small for gestational age (SGA) infants as a surrogate for FGR. The functional consequences of reduced BGN and the downstream targets of BGN were determined. Furthermore, the expression of targets was validated in primary placental endothelial cells isolated from FGR or control pregnancies. Approach and Results— BGN expression was determined using real-time polymerase chain reaction in placental tissues collected during chorionic villous sampling performed at 10 to 12 weeks’ gestation from pregnancies that had known clinical outcomes, including SGA. Short-interference RNA reduced BGN expression in telomerase-immortalized microvascular endothelial cells, and the effect on proliferation, angiogenesis, and thrombin generation was determined. An angiogenesis array identified downstream targets of BGN, and their expression in control and FGR primary placental endothelial cells was validated using real-time polymerase chain reaction. Reduced BGN expression was observed in SGA placental tissues. BGN reduction decreased network formation of telomerase-immortalized microvascular endothelial cells but did not affect thrombin generation or cellular proliferation. The array identified target genes, which were further validated: angiopoetin 4 (ANGPT4), platelet-derived growth factor receptor &agr; (PDGFRA), tumor necrosis factor superfamily member 15 (TNFSF15), angiogenin (ANG), serpin family C member 1 (SERPIN1), angiopoietin 2 (ANGPT2), and CXC motif chemokine 12 (CXCL12) in telomerase-immortalized microvascular endothelial cells and primary placental endothelial cells obtained from control and FGR pregnancies. Conclusions— This study reports a temporal relationship between altered placental BGN expression and subsequent development of SGA. Reduction of BGN in vascular endothelial cells leads to disrupted network formation and alterations in the expression of genes involved in angiogenesis. Therefore, differential expression of these may contribute to aberrant angiogenesis in SGA pregnancies.