Unexplained intrauterine growth restriction of the fetus (IUGR) results from impaired placental development, frequently associated with maternal malperfusion. Some cases are complicated further by preeclampsia (PE؉IUGR). Here, we provide the first evidence that placental protein synthesis inhibition and endoplasmic reticulum (ER) stress play key roles in IUGR pathophysiology. Increased phosphorylation of eukaryotic initiation factor 2␣ suggests suppression of translation initiation in IUGR placentas, with a further increase in PE؉IUGR cases. Consequently, AKT levels were reduced at the protein, but not mRNA, level. Additionally, levels of other proteins in the AKT-mammalian target of rapamycin pathway were decreased, and there was associated dephosphorylation of 4E-binding protein 1 and activation of glycogen synthase kinase 3␤. Cyclin D1 and the eukaryotic initiation factor 2B epsilon subunit were also down-regulated, providing additional evidence for this placental phenotype. The central role of AKT signaling in placental growth regulation was confirmed in Akt1 null mice, which display IUGR. In addition, we demonstrated ultrastructural and molecular evidence of ER stress in human IUGR and PE؉IUGR placentas, providing a potential mechanism for eukaryotic initiation factor 2␣ phosphorylation. In confirmation, induction of low-grade ER stress in trophoblast-like cell lines reduced cellular proliferation. PE؉IUGR placentas showed elevated ER stress with the additional expression of the pro-apoptotic protein C/EBP-homologous protein/growth arrest and DNA damage 153. These findings may account for the increased microparticulate placental debris in the maternal circulation of these cases, leading to endothelial cell activation and impairing placental development.

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.

Malperfusion of the placenta has been implicated as a cause of oxidative stress in complications of human pregnancy, leading to release of proinflammatory cytokines and anti-angiogenic factors into the maternal circulation. Uterine contractions during labor are known to be associated with intermittent utero-placental perfusion. We therefore tested whether oxidative stress, proinflammatory cytokines, and angiogenic regulators were increased in placentas subjected to short (<5 hours) and long (>15 hours) labor compared with nonlabored controls delivered by cesarean section. In addition, broader changes in gene transcripts were assessed by microarray analysis. Oxidative stress, activation of the nuclear factor-kappaB pathway, tumor necrosis factor-alpha and interleukin 1beta all increased in placental tissues after labor. Stabilization of hypoxia-inducible factor-1alpha and increased vascular endothelial growth factor soluble receptor-1 were also observed. By contrast, tissue levels of placenta growth factor decreased. Apoptosis was also activated in labored placentas. The magnitude of these changes related to the duration of labor. After labor, 55 gene transcripts were up-regulated and 35 down-regulated, and many of these changes were reflected at the protein level. In conclusion, labor is a powerful inducer of placental oxidative stress, inflammatory cytokines, and angiogenic regulators. Our findings are consistent with intermittent perfusion being the initiating cause. Placentas subjected to labor do not reflect the normal in vivo state at the molecular level.

Oxidative stress is central to ischemia-reperfusion injury. The role of the endoplasmic reticulum (ER) in this process is uncertain. In ER signaling, PERK-Nrf2 and Ire-CHOP are two pathways that determine cell fate under stress. PERK-Nrf2 up-regulates antioxidant enzyme expression whereas Ire-CHOP promotes apoptosis. We have identified a novel pathway in ER stress-induced apoptosis after ischemia-reperfusion in vitro involving translational suppression of the survival kinase PKB/ Akt (Akt), and elucidated an alternative protective role of antioxidants in the regulation of Akt activity. Using human choriocarcinoma JEG-3 cells, we found that sustained activation of ER stress by tunicamycin or thapsigargin exacerbated apoptosis in oxygen-glucose-deprived cells during reoxygenation. This was mediated via a reduction in phosphorylated Akt secondary to downregulation of protein translation rather than suppression of phosphorylation. Transient overexpression of wild-type Akt, but not kinase-dead Akt, in JEG-3 cells diminished tunicamycin-OGD reoxygenation-induced apoptosis. The antioxidants Trolox and Edaravone reduced apoptosis, but the protective effect of Trolox was abrogated by the PI3K inhibitor, LY294002. We speculate that sustained ER stress may contribute to the placental dysfunction seen in human pregnancy complications.-Yung, H-w., Korolchuk, S., Tolkovsky, A. M., Charnock-Jones, D. S., Burton, G. J. Endoplasmic reticulum stress exacerbates ischemia-reperfusion-induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells. Keywordsunfolded protein response; placenta; oxidative stress; trophoblast; CHOP PLACENTAL OXIDATIVE STRESS has been postulated to be a key factor in the pathogenesis of human pregnancy complications such as intrauterine growth retardation and pre-eclampsia (1,2). We recently proposed that the cause of the stress is an ischemia-reperfusion type injury (3). Unlike in other organs, where ischemia-reperfusion injury is usually an isolated insult caused by pathological blockage or rupture of blood vessels attenuating oxygen and nutrient supply, in the placenta mild ischemia-reperfusion is likely to be a repetitive process. Maternal blood flow through the intervillous space of the placenta may fluctuate through three principal mechanisms: intrinsic contraction of the spiral arteries supplying the placenta, external 1Correspondence:

Endoplasmic reticulum (ER) stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473) confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308). The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

Based on gestational age at diagnosis and/or delivery, pre-eclampsia (PE) is commonly divided into early-onset (<34 weeks) and late-onset (≥34 weeks) forms. Recently, the distinction between ‘placental’ and ‘maternal’ causation has been proposed, with ‘placental’ cases being more frequently associated with early-onset and intrauterine growth restriction. To test whether molecular placental pathology varies according to clinical presentation, we investigated stress-signalling pathways, including unfolded protein response (UPR) pathways, MAPK stress pathways, heat-shock proteins and AMPKα in placentae delivered by caesarean section for clinical indications at different gestational ages. Controls included second-trimester, pre-term and normal-term placentae. BeWo cells were used to investigate how these pathways react to different severities of hypoxia–reoxygenation (H/R) and pro-inflammatory cytokines. Activation of placental UPR and stress-response pathways, including P-IRE1α, ATF6, XBP-1, GRP78 and GRP94, P-p38/p38 and HSP70, was higher in early-onset PE than in both late-onset PE and normotensive controls (NTCs), with a clear inflection around 34 weeks. Placentae from ≥ 34 weeks PE and NTC were indistinguishable. Levels of UPR signalling were similar between second-trimester and term controls, but were significantly higher in pre-term ‘controls’ delivered vaginally for chorioamnionitis and other conditions. Severe H/R (1/20% O2) induced equivalent activation of UPR pathways, including P-eIF2α, ATF6, P-IRE1α, GRP78 and GRP94, in BeWo cells. By contrast, the pro-inflammatory cytokines TNFα and IL-1β induced only mild activation of P-eIF2α and GRP78. AKT, a central regulator of cell proliferation, was reduced in the < 34 weeks PE placentae and severe H/R-treated cells, but not in other conditions. These findings provide the first molecular evidence that placental stress may contribute to the pathophysiology of early-onset pre-eclampsia, whereas that is unlikely to be the case in the late-onset form of the syndrome. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Pregnancy at high altitude is associated with a reduction in birth weight of ∼100 g/1000 m of ascent. The underlying mechanisms are unclear but may involve alteration in energy-demanding activities, such as protein synthesis. To test this hypothesis, both in vivo and in vitro approaches were used. Placental tissues from pregnant women residing at 3100 m were studied, and placental cells were incubated under hypoxia. In the 3100-m placentas, we observed dilation of endoplasmic reticulum (ER) cisternae, increased phosphorylation of eukaryotic initiation factor 2 subunit α (P-eIF2α), reduced AKT phosphorylation, and reduced P-4E-BP1 but increased 4E-BP1 protein compared to sea level controls. These findings suggest the presence of ER stress and protein synthesis inhibition. Hypoxia (1% O2) reduced proliferation of trophoblast-like JEG-3 cells, BeWo cells, and placental fibroblasts by ∼40, ∼60, and ∼18%, respectively. Sublethal dosage of salubrinal, an eIF2α phosphatase inhibitor, increased P-eIF2α and reduced BeWo cell and placental fibroblast proliferation by ∼50%. Administration of the PI-3K inhibitor LY294002 also reduced JEG-3 proliferation. Our results demonstrate that exposure to chronic hypobaric hypoxia causes mild placental ER stress, which, in turn, modulates protein synthesis and slows proliferation. These effects may account for the reduced placental villous volume, and contribute to the low birth weight that typifies high-altitude populations.—Yung, H. W., Cox, M., Tissot van Patot, M., Burton, G. J. Evidence of endoplasmic reticulum stress and protein synthesis inhibition in the placenta of non-native women at high altitude.

Recent data have provided molecular evidence of high levels of endoplasmic reticulum stress in non-laboured placentas from cases of early-onset pre-eclampsia. Endoplasmic reticulum stress is intricately linked to oxidative stress, and the two often share the same aetiology. In the case of pre-eclampsia this is likely to be placental malperfusion, secondary to deficient conversion of the spiral arteries. Endoplasmic reticulum stress activates a number of signalling pathways aimed at restoring homeostasis, but if these attempts fail then the apoptotic machinery may be activated. The potential consequences for placental development and function are numerous and diverse. Inhibition of protein synthesis results in lower levels of many kinases, growth factors and regulatory proteins involved in cell cycle control, and experiments in vitro reveal that endoplasmic reticulum stress slows cell proliferation. Chronic, low levels of stress during the second and third trimesters may therefore result in a growth restricted phenotype. Higher levels of endoplasmic reticulum stress lead to activation of pro-inflammatory pathways, a feature of pre-eclampsia that may contribute to maternal endothelial cell activation. These findings emphasise the complexity of cellular responses to stress, and the need to approach these in a holistic fashion when considering therapeutic interventions.