Tereza Cindrova-Davies

Placental Endoplasmic Reticulum Stress and Oxidative Stress in the Pathophysiology of Unexplained Intrauterine Growth Restriction and Early Onset Preeclampsia

The pregnancy complications of unexplained intrauterine growth restriction and early onset preeclampsia are thought to share a common aetiology in placental malperfusion secondary to deficient maternal spiral artery conversion. A key question is whether the contrasting clinical manifestations reflect different placental pathologies, or whether they are due to altered maternal responses to a common factor derived from the placenta. Recently, molecular evidence of protein synthesis inhibition secondary to endoplasmic reticulum stress has provided an explanation for the small placental phenotype in both conditions. However, other pathways activated by more severe endoplasmic reticulum stress are only observed in placentas from pregnancies associated with early onset preeclampsia. Here, we review the literature and conclude that there is evidence of greater maternal vascular compromise of the placenta in these cases. We speculate that in cases of normotensive intrauterine growth restriction the placental pathology is centred predominantly around endoplasmic reticulum stress, whereas in cases complicated by preeclampsia oxidative stress is further superimposed. This causes the release of a potent mix of pro-inflammatory cytokines, anti-angiogenic factors and trophoblastic aponecrotic debris into the maternal circulation that causes the peripheral syndrome. Maternal and fetal constitutional factors may modulate how the placenta responds to the maternal vascular insult, and how the mother is affected by the placental factors released. However, the principal conclusion is that the difference between these two conditions lies in the severity of the initiating deficit in spiral arterial conversion, and the relative degrees of endoplasmic reticulum stress and oxidative stress induced in the placenta as a result.

Endometrial glands as a source of nutrients, growth factors and cytokines during the first trimester of human pregnancy: A morphological and immunohistochemical study

BackgroundThe maternal circulation to the human placenta is not fully established until 10–12 weeks of pregnancy. During the first trimester the intervillous space is filled by a clear fluid, in part derived from secretions from the endometrial glands via openings in the basal plate. The aim was to determine the activity of the glands throughout the first trimester, and to identify components of the secretions.MethodsSamples of human decidua basalis from 5–14 weeks gestational age were examined by transmission electron microscopy and immunohistochemically. An archival collection of placenta-in-situ samples was also reviewed.ResultsThe thickness of the endometrium beneath the implantation site reduced from approximately 5 mm at 6 weeks to 1 mm at 14 weeks of gestation. The glandular epithelium also transformed from tall columnar cells, packed with secretory organelles, to a low cuboidal layer over this period. The lumens of the glands were always filled with precipitated secretions, and communications with the intervillous space could be traced until at least 10 weeks. The glandular epithelium reacted strongly for leukaemia inhibitory factor, vascular endothelial growth factor, epidermal growth factor, transforming growth factor beta, alpha tocopherol transfer protein, MUC-1 and glycodelin, and weakly for lactoferrin. As gestation advanced uterine natural killer cells became closely approximated to the basal surface of the epithelium. These cells were also immunopositive for epidermal growth factor.ConclusionsMorphologically the endometrial glands are best developed and most active during early human pregnancy. The glands gradually regress over the first trimester, but still communicate with the intervillous space until at least 10 weeks. Hence, they could provide an important source of nutrients, growth factors and cytokines for the feto-placental unit. The endometrium may therefore play a greater role in regulating placental growth and differentiation post-implantation than previously appreciated.

Gabor Than Award Lecture 2008: Pre-eclampsia – From Placental Oxidative Stress to Maternal Endothelial Dysfunction

Pre-eclampsia is the most important complication of human pregnancy worldwide and a major contributor to maternal and fetal morbidity and mortality. Strong evidence exists that generation of placental oxidative stress, secondary to deficient spiral artery remodelling, is a key intermediary event, triggering the secretion of a mixture of placental factors that culminate in an enhanced maternal inflammatory response. Reactive oxygen species (ROS) have been recognised as secondary messengers in intracellular signalling cascades. Experiments studying placental ischaemia-reperfusion in vitro or in vivo during labour provide strong evidence suggesting that oxidative stress and ROS production can activate downstream stress-signalling pathways, p38 and SAPK/JNK MAPK, and the pro-inflammatory NF-kappaB signalling pathway, culminating in the release of inflammatory mediators, apoptotic debris, anti-angiogenic factors and other mediators, which then stimulate a maternal inflammatory reaction that manifests in endothelial dysfunction and the symptoms of pre-eclampsia. Addition of anti-oxidants or blocking the stress or inflammatory pathways in vitro attenuates these effects and opens possibilities for therapeutic intervention.

Soluble FLT1 sensitizes endothelial cells to inflammatory cytokines by antagonizing VEGF receptor-mediated signalling

AIMS Pre-eclampsia affects 5-7% of pregnancies, and is a major cause of maternal and foetal death. Elevated serum levels of placentally derived splice variants of the vascular endothelial growth factor (VEGF) receptor, soluble fms-like tyrosine kinase-1 (sFLT1), are strongly implicated in the pathogenesis but, as yet, no underlying mechanism has been described. An excessive inflammatory-like response is thought to contribute to the maternal endothelial cell dysfunction that characterizes pre-eclampsia. We hypothesized that sFLT1 antagonizes autocrine VEGF-A signalling, rendering endothelial cells more sensitive to pro-inflammatory factors also released by the placenta. We tested this by manipulating VEGF receptor signalling and treating endothelial cells with low doses of tumour necrosis factor-α (TNF-α). METHODS AND RESULTS Application of recombinant sFLT1 alone did not activate human umbilical vein endothelial cells (HUVECs). However, antagonizing the autocrine actions of endothelial VEGF-A and/or placenta growth factor (PlGF) by pre-incubation with recombinant sFLT1, anti-FLT1, anti-VEGF receptor 2 (KDR), anti-VEGF-A, VEGF receptor tyrosine kinase inhibitor SU5614, or knocking-down FLT1 or KDR transcripts rendered cells more sensitive to low doses of TNF-α. Each treatment increased activation, as measured by increases in endothelial intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), endothelin 1 (ET-1), von Willebrand factor (vWF), and leucocyte adhesion, and led to reduction in AKT Ser⁴⁷³ and endothelial nitric oxide synthase (eNOS) Ser¹¹⁷⁷ phosphorylation. CONCLUSIONS Our data describe a mechanism by which sFLT1 sensitizes endothelial cells to pro-inflammatory factors, providing an explanation for how placental stress may precipitate the pre-eclamptic syndrome.

Reduced Cystathionine γ-Lyase and Increased miR-21 Expression Are Associated with Increased Vascular Resistance in Growth-Restricted Pregnancies: Hydrogen Sulfide as a Placental Vasodilator

Increased vascular impedance in the fetoplacental circulation is associated with fetal hypoxia and growth restriction. We sought to investigate the role of hydrogen sulfide (H2S) in regulating vasomotor tone in the fetoplacental vasculature. H2S is produced endogenously by catalytic activity of cystathionine β-synthase and cystathionine γ-lyase (CSE). Immunohistochemical analysis localized CSE to smooth muscle cells encircling arteries in stem villi. Immunoreactivity was reduced in placentas from pregnancies with severe early-onset growth-restriction and preeclampsia displaying abnormal umbilical artery Doppler waveforms compared with preeclamptic placentas with normal waveforms and controls. These findings were confirmed at the protein and mRNA levels. MicroRNA-21, which negatively regulates CSE expression, was increased in placentas with abnormal Doppler waveforms. Exposure of villus explants to hypoxia-reoxygenation significantly reduced CSE protein and mRNA and increased microRNA-21 expression. No changes were observed in cystathionine β-synthase expression, immunolocalized principally to the trophoblast, in pathologic placentas or in vitro. Finally, perfusion of normal placentas with an H2S donor, after preconstriction with a thromboxane mimetic, resulted in dose-dependent vasorelaxation. Glibenclamide and NG-nitro-l-arginine methyl ester partially blocked the effect, indicating that H2S acts through ATP-sensitive K+ channels and nitric oxide synthesis. These results demonstrate that H2S is a powerful vasodilator of the placental vasculature and that expression of CSE is reduced in placentas associated with increased vascular resistance.

Human placental metabolic adaptation to chronic hypoxia, high altitude: hypoxic preconditioning

We have previously demonstrated placentas from laboring deliveries at high altitude have lower binding of hypoxia-inducible transcription factor (HIF) to DNA than those from low altitude. It has recently been reported that labor causes oxidative stress in placentas, likely due to ischemic hypoxic insult. We hypothesized that placentas of high-altitude residents acquired resistance, in the course of their development, to oxidative stress during labor. Full-thickness placental tissue biopsies were collected from laboring vaginal and nonlaboring cesarean-section term (37–41 wk) deliveries from healthy pregnancies at sea level and at 3,100 m. After freezing in liquid nitrogen within 5 min of delivery, we quantified hydrophilic and lipid metabolites using 31P and 1H NMR metabolomics. Metabolic markers of oxidative stress, increased glycolysis, and free amino acids were present in placentas following labor at sea level, but not at 3,100 m. In contrast, at 3,100 m, the placentas were characterized by the presence of concentrations of stored energy potential (phosphocreatine), antioxidants, and low free amino acid concentrations. Placentas from pregnancies at sea level subjected to labor display evidence of oxidative stress. However, laboring placentas at 3,100 m have little or no oxidative stress at the time of delivery, suggesting greater resistance to ischemia-reperfusion. We postulate that hypoxic preconditioning might occur in placentas that develop at high altitude.

The natural compound n-butylidenephthalide derived from the volatile oil of Radix Angelica sinensis inhibits angiogenesis in vitro and in vivo.

Radix Angelica sinensis is a Chinese medicinal herb that has been used extensively in the East for the treatment of cardiovascular diseases (CVDs). Angiogenesis plays an important role in the pathogenesis of CVDs. We hypothesized that Radix A. sinensis may contain angiogenesis modulators. In the current study, we investigated the effects of a volatile oil of Radix A. sinensis (VOAS) and n-butylidenephthalide (BP), one of the bioactive components in VOAS, on angiogenesis in vitro and in vivo. The results suggested that VOAS exerted anti-angiogenic effects by inhibiting human umbilical vein endothelial cell proliferation, migration and capillary-like tube formation on Matrigel. BP was also shown to be anti-angiogenic and its mechanisms were through inhibition of cell cycle progression and induction of apoptosis. Western blotting analysis indicated that the anti-angiogenic actions of BP were associated with the activation of p38 and ERK 1/2 but not SAPK/JNK and Akt signaling pathways. Further investigations showed that BP inhibited endothelial sprouting in an ex vivo mouse aortic ring model and was a potent inhibitor of the development of zebrafish subintestinal vessels in vivo. Our data using the volatile oil contrast with previous findings, which showed an aqueous extract of Radix A. sinensis was pro-angiogenic. This highlights the importance of identifying pro- and anti-angiogenic substances in Radix A. sinensis, not only for the development of novel angiogenesis modulators for the treatment of CVDs, but also to ensure the proper use of Radix A. sinensis as a nutraceutical.

Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats.

•  High‐altitude pregnancy is associated with reduced oxygenation and placental complications, which can affect maternal and fetal outcome. However, most high‐altitude populations are also impoverished and because maternal undernutrition itself is known to promote placental problems, the extent to which complications during high‐altitude pregnancy could be due to maternal oxygen and/or nutrient restriction remains unclear. •  The aim of the study was to investigate whether reduced placental oxygenation, independent of maternal undernutrition, increases maternal and placental oxidative stress and whether maternal treatment with vitamin C is protective. •  The study shows that hypoxic pregnancy increased maternal circulating and placental molecular indices of oxidative stress. •  Maternal vitamin C treatment was protective and increased birth weight. •  The study offers insight to mechanism and intervention against the effects of high altitude on pregnancy.

Evidence for Transcriptional Activity in the Syncytiotrophoblast of the Human Placenta

The aim was to test for evidence of transcriptional activity within the nuclei of the syncytiotrophoblast of the human placenta. The syncytiotrophoblast forms the epithelial covering of the villous tree, and is a multinucleated, terminally-differentiated syncytium generated through fusion of the underlying progenitor cytotrophoblast cells. Its nuclei are heterogeneous with respect to chromatin condensation, and previous functional studies of 3H-uridine uptake in vitro have indicated that they are transcriptionally inactive. This observation is surprising given the key roles this tissue plays in active transport, hormone synthesis and metabolic regulation, and has widespread implications for trophoblast physiology and pathophysiology. We used three different approaches to look for evidence of transcriptional activity. First, immunofluorescence staining was performed on paraffin-embedded early pregnancy and term placental villi, using an antibody directed specifically against the actively transcribing form of RNA polymerase II. Second, a nucleoside incorporation assay was applied to placental villi maintained in short-term culture, with and without the transcription blocker α-amanitin. Third, histone modifications associated with active chromatin were identified by immunohistochemistry and immunofluorescence. Each of these methods showed transcription to be occurring in a proportion of syncytiotrophoblast nuclei, with qualitative evidence for transcription being more abundant in the first trimester than at term. These findings correlated with electron microscopical observations of prominent nucleoli within the nuclei, particularly during early pregnancy, signifying transcription of ribosomal RNA. Contrary to previous findings, these results confirm that a proportion of syncytiotrophoblast nuclei actively produce mRNA transcripts.

The therapeutic potential of antioxidants, ER chaperones, NO and H2S donors, and statins for treatment of preeclampsia

Preeclampsia is a complex multifactorial disease. Placental oxidative stress, a result of deficient spiral artery remodeling, plays an important role in the pathophysiology of preeclampsia. Antiangiogenic factors secreted from malperfused placenta are instrumental in mediating maternal endothelial dysfunction and consequent symptoms of preeclampsia; the mechanism is likely to involve increased ET-1 secretion and reduced NO bioavailability. Therapeutic interventions so far remain only experimental and there is no established remedy for the treatment of preeclampsia. This review concentrates on the evidence for the therapeutic potential of antioxidants, ER chaperones, NO and H2S donors, and statins. These compounds display pleitropic antioxidant, anti-inflammatory, and pro-angiogenic effects in animal and in vitro studies. Although clinical trials on the use of antioxidant vitamins in pregnancy proved largely unsuccessful, the scope for their use still exists given the beneficial cardioprotective effects of antioxidant-rich Mediterranean diet, periconceptual vitamin use and the synergistic effect of vitamin C and L-arginine. Encouraging clinical evidence exists for the use of NO donors, and a clinical trial is underway testing the effect of statins in treatment of preeclampsia. H2S recently emerged as a novel therapeutic agent for cardiovascular disease, and its beneficial effects were also tested in animal models of preeclampsia. It is risky to prescribe any medication to pregnant women on a large scale, and any future therapeutic intervention has to be well tested and safe. Many of the compounds discussed could be potential candidates.