Placental Angiogenesis Research Articles

Effect of glucocorticoids on mechanisms of placental angiogenesis

The benefits of antenatal glucocorticoid (GC) treatment to promote human fetal lung maturation are well established. However, reports have emerged indicating that maternal exposure to high concentrations of circulating GCs alters placental and fetal development. Because many adult-onset metabolic and cardiovascular disorders have their origins in utero, the importance of prenatal conditions should be considered in detail. Therefore, this review aims to present an overview of the GC effect on placental and fetal development, specifically with regard to mechanisms of placental angiogenesis.We assumed that GC overexposure affects fetal development by altering placental angiogenesis. Disturbances in the development of the villous tree and pathological changes in the villous vascular system with insufficient uteroplacental blood flow have been linked to the pathogenesis of intrauterine growth retardation. Moreover, low birth weight is a serious risk factor known to correlate with an increased risk of adult-onset diseases. Although there have been many circumstances in which maternal GCs are elevated, we focused on exogenous synthetic GCs that are applied for therapeutic reasons. However, some questions about the use of steroids remain unanswered, which will require further studies that lead us to review alterations in placental angiogenesis under the perspective of GC overexposure.

Modulators of the Balance between M1 and M2 Macrophages during Pregnancy.

Macrophages are a subset of mononuclear phagocytes of the innate immune system with high plasticity and heterogeneity. At the maternal–fetal interface, macrophages are present in all stages of pregnancy and involved in a variety of activities, including regulation of immune cell activities, decidualization, placental cell invasion, angiogenesis, parturition, and postpartum uterine involution. The activation state and function of uterine–placental macrophages are largely dependent on the local tissue microenvironment. However, disruption of the uterine microenvironment can have profound effects on macrophage activity and subsequently impact pregnancy outcome. Thus, appropriately and timely regulated macrophage polarization has been considered a key determinant of successful pregnancy. Targeting macrophage polarization might be an efficient strategy for maintaining maternal–fetal immune homeostasis and a normal pregnancy. Here, we will review the latest findings regarding the modulators regulating macrophage polarization in healthy pregnancies and pregnancy complications, which might provide a basis for macrophage-centered therapeutic strategies.

Netrin-4 Expression and Regulation by Hypoxia in Human Placenta

Netrin-4 is a well-known actor involved in axonal guidance. Recently we have reported the cellular localization of netrin-4 in human placenta. Furthermore we demonstrated its role in placental angiogenesis. Here we determined netrin-4 mRNA expression in first trimester placenta. First, we showed an increase in netrin-4 mRNA expression at 11-13 weeks of gestation (wg), suggesting a negative regulation of netrin-4 expression by hypoxia. In fact, the human placenta undergoes a transition from a low oxygenated to a highly oxygenated environment during the first trimester of pregnancy. This physiological switch in oxygen tension is a prerequisite for proper placental development and involves the hypoxia-inducible factor (HIF-1), a protein that is up-regulated under hypoxic conditions. Then, we used the trophoblast cell line BeWo to test this hypothesis. BeWo cells cultured under hypoxic conditions exhibited decreased levels of netrin-4 mRNA expression. Using siRNA strategy, hypoxia-inducible factor 1α (HIF-1α) knock-down induced a significant increase in netrin-4 mRNA expression. Altogether these results indicate that netrin-4 expression in the trophoblast cells is regulated by hypoxia via the HIF-1 pathway.

877: Sildenafil treatment of non-severe hypertension during pregnancy alters placental protein expression involved in angiogenesis

Multiple proteins are involved in signaling pathways that regulate placental angiogenesis. We have previously described a murine model of non-severe hypertension (eNOS+/-) in which treatment with sildenafil (SIL) during pregnancy lowered maternal blood pressure without reducing fetal growth. Our aim was to investigate a potential mechanism for these findings. A murine model of non-severe hypertension (eNOS+/-) was obtained by breeding females with severe hypertension (eNOS-/-) with wild-type (WT) males. On gestational day 1 (GD 1), pregnant dams were randomly assigned either to SIL or water for 3 weeks. Four groups were generated: WT, WT-SIL, eNOS+/- and eNOS+/-SIL. On GD 18, dams were sacrificed, placentas were weighed and expression of placental proteins involved in angiogenesis were studied, including nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), hypoia inducible factor-1 (HIF-1), angiotensin-2, heme oxygenase-1 (HO-1), platelet derived growth factor receptor (PDGF-R) and placental growth factor (PLGF). Placental weights were similar among all groups (P=0.71). Placental protein expression of VEGF was higher in the eNOS+/- (hypertension) group compared to the WT group. Treatment with SIL decreased VEGF expression to a level similar to WT placentas (P=0.04, Figure 1). iNOS expression was lower in the eNOS+/- group compared to WT controls; however, SIL treatment increased iNOS expression, similar to the WT group (eNOS+/-SIL 0.35±0.02 vs eNOS+/-0.47±0.04, P=0.04). PLGF was higher in the eNOS+/- group compared to WT controls and SIL decreased PLGF expression, similar to the WT placentas (P=0.01, Figure 2). HIF-1 was decreased in the eNOS+/- group compared to WT controls (eNOS+/- 0.01±0.01 vs WT 0.02±0.01, P=0.02), but no difference was observed after treatment with SIL. Angiotensisn-2 was also increased in the eNOS+/- group compared to WT controls (eNOS+/-0.52±0.11 vs WT 0.41±0.01, P=0.01), SIL did not alter its expression. HO-1 and PDGF-R were similar among groups. In a murine model of non-severe hypertension, alterations in iNOS, VEGF and PLGF expression were improved with SIL. These findings may explain how SIL treatment of non-severe hypertension during pregnancy improves maternal blood pressure without reducing fetal growthView Large Image Figure ViewerDownload Hi-res image Download (PPT)

MicroRNA-16 inhibits feto-maternal angiogenesis and causes recurrent spontaneous abortion by targeting vascular endothelial growth factor.

Recurrent spontaneous abortion (RSA) is a common health problem that affects women of reproductive age. Recent studies have indicated that microRNAs are important factors in miscarriage. This study investigated the role of miR-16 in regulating vascular endothelial growth factor (VEGF) expression and the pathogenesis of RSA. In this report, clinical samples revealed that miR-16 expression was significantly elevated in the villi and decidua of RSA patients. In vitro, miR-16 upregulation inhibited human umbilical vein endothelial cell proliferation, migration and tube formation. Conversely, the downregulation of miR-16 reversed these effects. In vivo, we demonstrated that abnormal miR-16 levels affect the weights of the placenta and embryo and the number of progeny and microvascular density, as well as cause recurrent abortions by controlling VEGF expression in pregnant mice. VEGF, a potential target gene of miR-16, was inversely correlated with miR-16 expression in the decidua of clinical samples. Furthermore, the luciferase reporter system demonstrated that miR-16 was found to directly downregulate the expression of VEGF by binding a specific sequence of its 3′-untranslated region (3′UTR). Collectively, these data strongly suggest that miR-16 regulates placental angiogenesis and development by targeting VEGF expression and is involved in the pathogenesis of RSA.

Fluid Shear Stress Promotes Placental Growth Factor Upregulation in Human Syncytiotrophoblast Through the cAMP-PKA Signaling Pathway.

The effects of fluid shear stress (FSS) on the human syncytiotrophoblast and its biological functions have never been studied. During pregnancy, the syncytiotrophoblast is the main source of placental growth factor (PlGF), a proangiogenic factor involved in the placental angiogenesis and the vascular adaptation to pregnancy. The role of FSS in regulating PlGF expression in syncytiotrophoblasts is unknown. We investigated the impact of FSS on the production and secretion of the PlGF by the human syncytiotrophoblasts in primary cell culture. Laminar and continuous FSS (1 dyn cm-2) was applied to human syncytiotrophoblasts cultured in a parallel-plate flow chambers. Secreted levels of PlGF, sFlt-1 (soluble fms-like tyrosin kinase-1), and prostaglandin E2 were tested by immunologic assay. PlGF levels of mRNA and intracellular protein were examined by RT-PCR and Western blot, respectively. Intracellular cAMP levels were examined by time-resolved fluorescence resonance energy transfer cAMP accumulation assay. Production of cAMP and PlGF secretion was significantly increased in FSS conditions compared with static conditions. Western blot analysis of cell extracts exposed to FSS showed an increased phosphorylation of protein kinase A substrates and cAMP response element-binding protein on serine 133. FSS-induced phosphorylation of cAMP response element-binding protein and upregulation of PlGF were prevented by inhibition of protein kinase A with H89 (3 μmol/L). FSS also triggers intracellular calcium flux, which increases the synthesis and release of prostaglandin E2. The enhanced intracellular cAMP in FSS conditions was blocked by COX1/COX2 (cyclooxygenase) inhibitors, suggesting that the increase in prostaglandin E2 production could activate the cAMP/protein kinase A pathway in an autocrine/paracrine fashion. FSS activates the cAMP/protein kinase A pathway leading to upregulation of PlGF in human syncytiotrophoblast.

Inhibition of Wnt Inhibitory Factor 1 Under Hypoxic Condition in Human Umbilical Vein Endothelial Cells Promoted Angiogenesis in Vitro.

Placentation is a complicated process critical for maternal-fetal exchange of nutrients and gases that includes stepwise vasculogenesis and angiogenesis. Wnt inhibitory factor 1 (WIF1) is a secreted Wnt antagonist that acts as a tumor-suppressor gene by antagonizing angiogenesis and proliferation and inducing apoptosis. The purpose of this study was to investigate the function of WIF1 on placental angiogenesis in human umbilical vein endothelial cells (HUVECs) under hypoxic conditions. We found that WIF1 was diversely expressed in placental vascular endothelial cells at different points during gestation and was weaker in the early placenta than in the term placenta. We validated the antiangiogenesis role of WIF1 by inhibiting proliferation, tube formation and migration, and inducing apoptosis of endothelial cells through antagonizing Wnt/β-catenin signaling pathway. We also identified that hypoxic conditions similar to the early placenta inhibited the expression of WIF1 and reversed the antiangiogenesis of WIF1 in HUVECs. In conclusion, our present study supported the hypothesis that WIF1 is crucial as a negative regulator of the functions of endothelial cells in angiogenesis and that hypoxia plays an important role in controlling WIF1 expression and angiogenesis. We also demonstrated that Wnt/β-catenin signaling pathway was activated in correspondence with the suppression of WIF1 in the angiogenesis of endothelial cells under hypoxic conditions.

Maternal citrulline supplementation enhances placental function and fetal growth in a rat model of IUGR: involvement of insulin-like growth factor 2 and angiogenic factors

Objective: To determine the effects of maternal citrulline supplementation on fetal growth and placental efficiency in a rat model of intrauterine growth restriction (IUGR) induced by maternal protein restriction.Methods: Pregnant Sprague–Dawley rats were randomly assigned to three groups: NP (receiving a control 20% protein diet), LP (a 4% protein diet), or LP-CIT (an LP diet along with L-citrulline, 2 g/kg/d in drinking water). On the 15th and 21st day of gestation (GD15 and GD21, respectively), dams underwent a C-section, by which fetuses and placentas were extracted. The expression of genes involved in placental growth and angiogenesis was studied by quantitative RT-PCR.Results: Maternal citrulline supplementation increased fetal weight at GD21, and fetal weight/placental weight ratio, an index of placental efficiency, from mid gestation (p < 0.001). The expression of Igf2-P0, a placenta-specific variant of insulin-like growth factor 2 (Igf2) gene, and Vegf and Flt-1, involved in angiogenic pathways, was enhanced in the LP-CIT group (versus NP, p < 0.001, p < 0.01, and p < 0.05 for Igf2-P0, Vegf, and Flt-1, respectively).Conclusions: In a model of IUGR induced by protein deprivation, citrulline enhances fetal growth, placental efficiency, and the expression of genes involved in angiogenesis. The relevance of such effect in human pregnancies complicated by IUGR warrants further study.

Expressions of VEGF-A and VEGFR-2 in placentae from GDM pregnancies.

BackgroundGestational diabetes mellitus (GDM) is one of the most common medical complications of pregnancy, and has important health implications for mother and child. Changes in the fetoplacental vessels may predict those in the vasculature of the developing fetus, as these have been implicated in the pathogenesis of human GDM. This study aimed to determine the differences in the localization and expression level of VEGFA and VEGFR2 between placentas of women with GDM and placentas of normal pregnancies, which is the first step in elucidating the possible roles of VEGFA and VEGFR2 in the altered uteroplacental function resulting from maternal hyperglycaemia and ultimately in the manifestation of GDM.MethodsThe expressions of VEGFA and VEGFR2 mRNA and protein in 20 samples from each group were analyzed by real-time PCR, immunohistochemistry and Western blot. The placental blood barrier and angiogenesis were observed by the transmission electron microscopy (TEM) in10 GDM samples and ten controls.ResultsThe expression levels of VEGFA and VEGFR2 mRNA and protein were significantly decreased in the GDM group (P < 0.05 or 0.01). Immunohistochemical analysis showed the reduced expression of VEGFA and VEGFR2 protein in GDM-affected placental tissues, and the degenerative alterations of the terminal villi vascular.ConclusionThe expressions of VEGFA and VEGFR-2 mRNAs and protein were reduced in GDM-affected placental tissues, suggesting that maternal GDM affects the pathophysiological function of placentas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0191-8) contains supplementary material, which is available to authorized users.

Subclinical Pregnancy Toxemia-Induced Gene Expression Changes in Ovine Placenta and Uterus.

The objective was to elucidate gene expression differences in uterus, caruncle, and cotyledon of ewes with subclinical pregnancy toxemia (SCPT) and healthy ewes, and to identify associated biological functions and pathways involved in pregnancy toxemia. On Day 136 (±1 day) post-breeding, ewes (n = 18) had body condition score (BCS; 1–5; 1, emaciated; 5, obese) assessed, and blood samples were collected for plasma glucose and β-hydroxybutyrate (BHBA) analyses. The ewes were euthanized, and tissue samples were collected from the gravid uterus and placentomes. Based on BCS (2.0 ± 0.02), glucose (2.4 ± 0.33), and BHBA (0.97 ± 0.06) concentrations, ewes (n = 10) were grouped as healthy (n = 5) and subclinical SCPT (n = 5) ewes. The mRNA expressions were determined by quantitative PCR method, and prediction of miRNA partners and target genes for the predicted miRNA were identified using miRDB (http://mirdb.org/miRDB/). Top ranked target genes were used to identify associated biological functions and pathways in response to SPCT using PANTHER. The angiogenesis genes VEGF and PlGF, and AdipoQ, AdipoR2, PPARG, LEP, IGF1, IGF2, IL1b, and TNFα mRNA expressions were lower in abundances, whereas hypoxia genes eNOS, HIF1a, and HIF 2a, and sFlt1 and KDR mRNA expressions were greater in abundances in uterus and placenta of SCPT ewes compared to healthy ewes (P < 0.05). The predicted miRNA and associated target genes contributed to several biological processes, including apoptosis, biological adhesion, biological regulation, cellular component biogenesis, cellular process, developmental process, immune system process, localization, metabolic process, multicellular organismal process, reproduction, and response to stimulus. The target genes were involved in several pathways including angiogenesis, cytoskeletal regulation, hypoxia response via HIF activation, interleukin signaling, ubiquitin proteasome, and VEGF signaling pathway. In conclusion, genes associated with blood vessel remodeling were lower in abundances and that the genes associated with hypoxic conditions were greater in abundances in the uteroplacental compartment of SCPT ewes. It is obvious that the factors that influence placental vascular development and angiogenesis as noted in this study set the course for hemodynamic changes and hence have a major impact on the rate of transplacental nutrient exchange, fetal growth, and health of the dam.

Modulation of factors involved in placental haemostasis and angiogenesis by low-molecular-weight-heparins.

In placentae from uneventful pregnancies a direct relationship between expression of tissue factor (TF) and tissue-factor pathway inhibitor type 2 (TFPI2) was found, as well as between TF and vascular endothelial growth factor (VEGF). Furthermore, placentae from gestational vascular complications (GVCs) lack these correlations. Aims of the present study are (1) to evaluate a possible role of low-molecular-weight-heparins (LMWHs) in the modulation of the expression of TF, TFPI, TFPI2 and VEGF in placentae from thrombophilic women and (2) to study the possible role of endothelium in the placental expression of markers involved in haemostasis and angiogenesis. Fourteen pregnancies in thrombophilic women and 11 uneventful pregnancies in non-thrombophilic women were studied and placentae collected. From each placenta total RNA was obtained. Expression of TF, TFPI, TFPI2 and VEGF was evaluated. Human Vein Endothelial Cells were incubated with increasing doses of LMWH and expression of TF, TFPI and VEGF was measured. Expression of all the markers analyzed in placentae from treated pregnancies was similar to that observed in placentae from uneventful ones. A significant direct relationship between TF and TFPI2, as well as TF and VEGF, was observed in cases treated with LMWHs and controls. Furthermore, the expression of TF and its inhibitors and VEGF in endothelial cells was modulated by LMWH. Present data suggest that LMWH during pregnancy in thrombophilic women restores the relationship between markers of haemostasis and angiogenesis. Furthermore, the endothelium is likely to play an important role in this phenomenon.

Placental vascularity and markers of angiogenesis in relation to prenatal growth status in overnourished adolescent ewes

IntroductionPlacental vascularity may be important in the development of fetal growth restriction (FGR). The overnourished adolescent ewe is a robust model of the condition, with ∼50% of offspring demonstrating FGR (birthweight >2 standard deviations below optimally-fed control mean). We studied whether placental vascularity, angiogenesis and glucose transport reflect FGR severity. MethodsSingleton pregnancies were established in adolescent ewes either overnourished to putatively restrict fetoplacental growth (n = 27) or control-fed (n = 12). At 131d (term = 145d) pregnancies were interrupted and fetuses classified as FGR (n = 17, <4222 g, -2SD below control-fed mean) or non-FGR (n = 10). Placentome capillary area density (CAD), number density (CND), surface density (CSD), and area per capillary (APC) in the fetal cotyledon (COT) and maternal caruncle (CAR) were analysed using immunostaining. COT/CAR mRNA expression of angiogenic ligands/receptors and glucose transporters were measured by qRT-PCR. ResultsFetal weight was reduced in FGR vs. Non-FGR/Control groups. Total placentome weight was Control > Non-FGR > FGR and fetal:placental weight ratios were higher in overnourished versus Control groups. COT vascular indices were Non-FGR > FGR > Control. COT-CAD, CSD and APC were significantly greater in Non-FGR overnourished versus Control and intermediate in FGR groups. CAR vascularity did not differ. CAR-VEGFA/FLT1/KDR/ANGPT1/ANGPT2/SLC2A1/SLC2A3 mRNA was lower and COT-ANGPT2 higher in overnourished versus Control groups. DiscussionRelative to control-intake pregnancy, overnourished pregnancies are characterised by higher COT vascularity, potentially a compensatory response to reduced nutrient supply, reflected by higher fetal:placental weight ratios. Compared with overnourished pregnancies where fetal growth is relatively preserved, overnourished pregnancies culminating in marked FGR have less placental vascularity, suggesting incomplete adaptation to the prenatal insult.

Homeobox gene TGIF-1 is increased in placental endothelial cells of human fetal growth restriction.

Aberrant placental angiogenesis is associated with fetal growth restriction (FGR). In mice, targeted disruption of the homeobox gene, transforming growth β-induced factor (Tgif-1), which is also a transcription factor, causes defective placental vascularisation. Nevertheless, the role of TGIF-1 in human placental angiogenesis is unclear. We have previously reported increased TGIF-1 expression in human FGR placentae and demonstrated localisation of TGIF-1 protein in placental endothelial cells (ECs). However, its functional role remains to be investigated. In this study, we aimed to specifically compare TGIF-1 mRNA expression in placental ECs isolated from human FGR-affected pregnancies with gestation-matched control pregnancies in two independent cohorts from Australia and Canada and to identify the functional role of TGIF-1 in placental angiogenesis using the human umbilical vein endothelial cell-derived cell line, SGHEC-7, and primary human umbilical vein ECs. Real-time PCR revealed that TGIF-1 mRNA expression was significantly increased in ECs isolated from FGR-affected placentae compared with that of controls. The functional roles of TGIF-1 were determined in ECs after TGIF-1 siRNA transfection. TGIF-1 inactivation in ECs significantly reduced TGIF-1 at both the mRNA and protein levels, as well as the proliferative and invasive potential, but significantly increased the angiogenic potential. Using angiogenesis PCR screening arrays, we identified ITGAV, NRP-1, ANPGT-1 and ANPGT-2 as novel downstream targets of TGIF-1, after TGIF-1 inactivation in ECs. Collectively, these results show that TGIF-1 regulates EC function and the expression of angiogenic molecules; and when abnormally expressed, may contribute to the aberrant placental angiogenesis observed in FGR.

131 Assessment of angiogenesis modulators in pregnant women with pre-eclampsia: A case-control study

Introduction Among the causes of complications in pregnancy and childbirth, hypertensive disorders are responsible for high maternal and fetal morbidity and mortality rates. Among them, preeclampsia(PE) deserves special mention, as it is a multisystem disorder of unknown aetiology, specific to human pregnancies, which exposes the mother and infant to serious risks, and which has an incidence ranging from 5% to 7% . The pathophysiology of PE is not yet fully understood because it presents a complex interaction of environmental, genetic and immunological factors. Objectives This study aimed to evaluate the serum concentration of factors associated with placental angiogenesis in pre-eclamptic and normotensive pregnant women. Methods This was a prospective, cross-sectional, case-control study in which the pro-angiogenic factors PlGF, VEGF, and IL-10, and the anti-angiogenic factors IL -6, IL-17, and TNF- α of 55 pregnant women (31 with pre-eclampsia and 24 normotensive), with gestational age ⩾ 20 weeks, were measured in maternal blood through the enzyme-linked immunosorbent assay (ELISA). The Mann–Whitney and Kruskal–Wallis tests were used for comparison between groups. Results Serum PIGF was reduced in the group of pregnant women with PE when compared with the normotensive women (493.2 ± 55.1 pg/mL vs. 4.4 ± 26.5 pg/mL; p 0.001 ). There was no significant difference in PlGF levels in the pre-eclamptic pregnant women in relation to gestational age or proteinuria levels ( p > 0.05 ). The serum levels of VEGF, IL-17, IL-10, and TNF- α were lower in the pregnant women with PE when compared with their normotensive peers, while the IL-6 levels were higher; however, this difference was not statistically significant ( p > 0.05 ). Conclusions Serum PlGF levels were reduced in the pregnant women with PE and were unrelated to disease severity. Serum levels of VEGF, IL-17, IL-10, and TNF- α were reduced in the pre-eclamptic pregnant women when compared with their normotensive peers, without statistically significant differences.

Total oxidative and anti-oxidative status, and ADAMTS-12 levels in placenta previa and early-onset severe preeclampsia.

This study was carried out due to the discussions in the literature stating that the inverse association between placenta previa (PP) and preeclampsia (PE). The aim of this study was to determine whether total antioxidant status (TAS), and total oxidant status (TOS) and ADAMTS-12 levels differ among early-onset (<34th gestational week) severe PE (EOS-PE), PP and uncomplicated pregnancies. In this case-control study, serum samples obtained from 26 pregnant with EOS-PE, 31 pregnant with PP, and 32 healthy patients with uncomplicated pregnancies (control group). TOS levels were significantly higher in the EOS-PE than in the control group and PP groups (p=0.002, p=0.05, respectively). TAS levels were significantly lower in the EOS-PE than in the control (p<0.001). Although TAS levels were lower in the EOS-PE group than in the PP group, the differences were not statistically significant (p=0.09). There were no significant differences in the ADAMTS-12 levels of the groups. The data in this study suggested that the balance between oxidative and anti-oxidative substances were comparable and normal in pregnancies complicated by PP when compared to normal pregnancies without placentation abnormality. In support of this, we encountered no case with PE and fetal growth restriction in our study groups suggesting normal placental angiogenesis. Contrarily, EOS-PE was associated with decreased TAS and increased TOS levels in the maternal serum.

First trimester trophoblasts forming endothelial-like tubes in vitro emulate a ‘blood vessel development’ gene expression profile

Extravillous cytotrophoblasts isolated from first trimester placenta, and immortalised cell lines derived from them, have the intrinsic ability to form endothelial-like tubes when cultured on Matrigel™ extracellular matrix. This in vitro tube formation may model placental angiogenesis and/or endovascular differentiation by trophoblasts. To interpret the relevance of this phenomenon to placental development, we used a gene expression microarray approach to identify which genes and pathways are associated with the tube-forming phenotype of HTR8/SVneo first trimester trophoblasts (HTR8-M), compared with HTR8/SVneo not forming tubes on plastic culture surface (HTR8-P). Furthermore, we used weighted gene co-expression network analysis (WGCNA) of microarray data to identify modules of co-expressed genes underlying the biological processes. There were 481 genes differentially expressed between HTR8-M and HTR8-P and these were significantly enriched for blood vessel development and related gene ontologies. WGCNA clustered the genes into 9 co-expression modules. One module was significantly associated with HTR8-M (p = 1.15E-05) and contained genes involved in actin cytoskeleton organization, cell migration and blood vessel development, consistent with tube formation on Matrigel. Another module was significantly associated with HTR8-P (p = 1.94E-05) and was enriched for genes involved in mitosis, consistent with proliferation by cells on plastic which do not differentiate. Up-regulation of angiogenesis and vascular development pathways in endovascular trophoblasts in vivo could underpin spiral artery remodelling processes, which are defective in preeclamptic pregnancies.

Expression of factors involved in the regulation of angiogenesis in the full-term human placenta: Effects of in vitro fertilization

The effects of assisted reproductive technologies (ARTs) on the safety of pregnancy and the resulting offspring remain controversial. Studies of placental functions, especially vasculogenesis and angiogenesis, in pregnancies established through ART are helpful for furthering our understanding of the safety of ART. This study compares the expression profiles of angiogenic factors in human term placentas obtained from natural (NAT) pregnancies vs. placentas obtained from pregnancies that resulted from ART. Term placentas were obtained from women who underwent an ART procedure (n=4), and these were compared with term placentas that were obtained from women who had experienced a spontaneous pregnancy (controls, n=4). An array analysis was performed using the Human Angiogenesis Antibody Array to detect 43 angiogenic factors and to identify which of these factors were differentially expressed between the two groups. The expression of six of these factors was greater in the ART group than in the NAT group. The levels of four of them, including vascular endothelial growth factor receptor-3 (VEGFR3), basic fibroblast growth factor (bFGF), interferon gamma (IFNG) and matrix metalloproteinase 1 (MMP1), were quantified using western blot analysis. These factors were examined using immunohistochemistry and microscopy in vascular endothelial cells or the cytoplasm and membranes of syncytiotrophoblast cells. Our finding that selected angiogenic factors exhibit altered expression profiles in ART placentas might be significant when evaluating ART safety.

Fetal blood vessel count increases in compensation of hypoxia in premature placentas

BACKGROUND Prematurity refers to live births before 37 weeks of gestation, wherein the baby is born before the body and its organ systems achieve perfect maturity, and this disorder is still a global problem. The high incidence of prematurity is a problem in developing and also in developed countries. Certain conditions accompanying pregnancies like preeclampsia, infection, and placental insufficiency, may trigger uterine hypoxia, causing premature birth. The placental condition is related to the intra-uterine fetal condition. In prolonged placental hypoxia, there occurs a compensatory mechanism, i.e. an increase in placental angiogenesis. This study aimed to evaluate the effect of hypoxia on fetal blood vessel count as compensatory mechanism for tissue hypoxia. METHODS An observational-analytical cross-sectional design using paraffin blocks of conserved premature placentas, comprising 31 samples of hypoxic premature placentas and 28 samples of non-hypoxic premature placentas, selected using non-random consecutive sampling. The samples were made into slides and stained with hematoxylin-eosin for assessment of histological structure, including fetal blood vessel count and integrity, villus conditions, syncytiotrophoblastic nuclear changes, and syncytiotrophoblastic nuclear aggregation. Mann-Whitney test was used to compare the difference of blood vessel count between groups. RESULTS Assessment of histological structure showed a significant increase in fetal blood vessel count in the hypoxic group [8.00 (5-15)] as compared with the non-hypoxic group [7.50 (3-15)]. CONCLUSION The hypoxia in premature placentas caused an increase in the number of fetal blood vessels as a form of compensation for disturbed oxygen homeostasis.

Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling

abstractThe objective of this study was to investigate whether human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit2 and endothelial cell Roundabout (Robo) receptors are involved in placental angiogenesis. The hPMSC-conditioned medium and human umbilical vein endothelial cells were studied for Slit2 and Robo receptor expression by immunoassay and RT-PCR. The effect of the conditioned medium of hPMSCs with or without Slit2 depletion on endothelial cells was investigated by in vitro angiogenesis using growth factor-reduced Matrigel. hPMSCs express Slit2 and both Robo1 and Robo4 are present in human umbilical vein endothelial cells. Human umbilical vein endothelial cells do not express Robo2 and Robo3. The hPMSC-conditioned medium and Slit2 recombinant protein significantly inhibit the endothelial cell migration, but not by the hPMSC-conditioned medium with Slit2 depletion. The hPMSC-conditioned medium and Slit2 significantly enhance endothelial tube formation with increased cumulated tube length, polygonal network number and vessel branching point number compared to endothelial cells alone. The tube formation is inhibited by the depletion of Slit2 from the conditioned medium, or following the expression of Robo1, Robo4, and both receptor knockdown using small interfering RNA. Furthermore, co-immunoprecipitation reveals Slit2 binds to Robo1 and Robo4. Robo1 interacts and forms a heterodimeric complex with Robo4. These results suggest the implication of both Robo receptors with Slit2 signaling, which is involved in endothelial cell angiogenesis. Slit2 in the conditioned medium of hPMSCs has functional effect on endothelial cells and may play a role in placental angiogenesis.

Genetic variation in vascular endothelial growth factor gene and its association with recurrent spontaneous abortion.

Vascular endothelial growth factor (VEGF) plays a main role in fetal and placental angiogenesis and is secreted by different cells of endometrium and placenta. In the present study we investigated the association of VEGF gene polymorphisms with recurrent spontaneous abortion (RSA). A case-control study of 100 women with at least two consecutive pregnancy losses before 20 weeks of gestational age and 100 fertile controls was performed to evaluate four VEGF gene polymorphisms including +936C/T (rs3025039), -154G>A (rs1570360), rs3025010 and +5092A/C (rs2146323). Genotyping was performed by PCR based restriction fragment length polymorphism (PCR-RFLP) analysis. Haplotype frequency was estimated for three SNPs' genotypes. Analysis of genetic STRUCTURE and K means clustering were performed to estimate genetic variation. We found an association between -154G/A heterozygous genotype (GA) and RSA. The VEGF single nucleotide polymorphism (SNP) in intron region (rs3025010) in different inheritance models was also associated with RSA. Linkage disequilibrium analysis revealed that VEGF SNP in intron 5 (rs3025010) was linked to promoter region SNP (rs1570360). Cluster analysis including Neighbor Joining and K-means clustering supported genetic differentiation of women with RSA and controls. Allelic polymorphisms in common VEGF SNPs was associated with RSA samples and haplotypes with at least one minor allele showing an association with RSA pathogenesis (Tab. 8, Fig. 2, Ref. 35).