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 α (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.

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.

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.

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.

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, 3ßHSD, ßhCG and syncytin. Therefore, we conclude that increased DLX3 expression in FGR may contribute to trophoblast dysfunction observed in FGR.