D. Stephen Charnock-Jones

Hypoxia-Reoxygenation A Potent Inducer of Apoptotic Changes in the Human Placenta and Possible Etiological Factor in Preeclampsia

Preeclampsia is a severe disorder of human pregnancy characterized by generalized activation of maternal endothelial cells. Oxidative stress of the placenta is considered a key intermediary step, precipitating deportation of apoptotic fragments into the maternal circulation, but the cause remains unknown. We hypothesize that intermittent placental perfusion, secondary to deficient trophoblast invasion of the endometrial arteries, leads to an ischemia-reperfusion–type insult. We therefore tested whether hypoxia-reoxygenation (H/R) in vitro stimulates apoptosis in human placental tissues compared with controls kept hypoxic or normoxic throughout. After H/R, release of cytochrome c from mitochondria was significantly increased and was associated with intense immunolabeling for active caspase 3 in the syncytiotrophoblast and fetal endothelial cells. There was also increased labeling of syncytiotrophoblastic nuclei for cleaved poly (ADP-ribose) polymerase (PARP), and higher cytosolic concentrations of cleaved PARP fragment were detected by Western blot. Syncytiotrophoblastic nuclei displayed increased chromatin condensation, and a significantly greater percentage was TUNEL positive. These changes were accompanied by increased lactate dehydrogenase release into the medium. Preadministration of the free radical scavenger, desferrioxamine, reduced cytochrome c release and the TUNEL-positive index, suggesting generation of hydroxyl radicals mediates these processes. By contrast, hypoxia alone caused a smaller increase in the TUNEL-positive index, and the majority of syncytiotrophoblastic nuclei displayed karyolysis, whereas normoxic controls remained euchromatic. We conclude that H/R stimulates apoptotic changes within the syncytiotrophoblast, whereas hypoxia principally induces necrosis. The quality of placental perfusion may therefore be a more important factor in the pathophysiology of preeclampsia than the absolute quantity.

Evidence of Placental Translation Inhibition and Endoplasmic Reticulum Stress in the Etiology of Human Intrauterine Growth Restriction

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 2alpha 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 3beta. 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 2alpha 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.

Redefining Preeclampsia Using Placenta-Derived Biomarkers

Preeclampsia affects 3% to 8% of all pregnancies.1 Acute maternal complications include eclampsia, stroke, placental abruption, disseminated intravascular coagulation, HELLP (hemolysis, elevated liver enzymes, low platelets), liver hemorrhage or rupture, pulmonary edema, adult respiratory distress syndrome, acute renal failure, and death.2 Preeclampsia complications account for more than 50 000 maternal deaths annually.2,3 In developing countries, where lack of access to appropriate maternal care is a major problem, maternal death rates are as high as 15% as compared with 0% to 1.8% in industrialized countries.2 Perinatal consequences include stillbirth, preterm delivery, fetal growth restriction (FGR), neonatal complications, and later sequelae.4 Long-term maternal risks include chronic hypertension, diabetes mellitus, coronary artery disease,5 neurological deficit, and premature death.2 Here, we argue that the classic definitions of preeclampsia, based on concepts that are now more than 50 years old, have become outdated and that the definition could be modernized to take account of our current understanding of disease pathophysiology. We propose a first step that incorporates the placental biomarker placenta growth factor (PlGF), but we allow for the possibility that the definition may need to be expanded to include other factors, such as the antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFLT1) or soluble endoglin (sENG),6 in due course. This is intended as an exploratory rather than a final development. Diseases may be defined and classified by cause, pathogenesis, or by clinical findings. Clear definitions and classification are difficult when pathogenesis is unknown. As a result, diagnostic labels may reflect only a set of symptoms and signs, defining a syndrome. Syndromes are never precise, because the features are multiple, nonspecific, and therefore may have diverse causes. Preeclampsia is a syndrome of new onset hypertension and proteinuria in the second half of pregnancy, that was defined …

Secretion of Tumor Necrosis Factor-α from Human Placental Tissues Induced by Hypoxia-Reoxygenation Causes Endothelial Cell Activation in Vitro: A Potential Mediator of the Inflammatory Response in Preeclampsia

Preeclampsia is a hypertensive complication of human pregnancy characterized by generalized maternal endothelial cell activation. Circulating pro-inflammatory cytokines derived from the placenta are thought to play a key role. We recently demonstrated that hypoxia-reoxygenation (H/R) of placental tissues in vitro causes equivalent oxidative stress to that seen in preeclampsia. Our aim was to determine whether H/R also increases production of tumor necrosis factor-alpha (TNF-alpha), and whether conditioned media from samples exposed to H/R causes activation of human umbilical vein endothelial cells (HUVECs). Concentrations of mRNA encoding TNF-alpha were significantly higher in placental tissues subjected to H/R compared to hypoxic or normoxic controls. Although there was no difference in the concentrations of TNF-alpha protein in tissue homogenates, levels of TNF-alpha protein in the medium were significantly higher after H/R compared to controls, indicating increased secretion. Furthermore, conditioned medium from samples subjected to H/R caused increased expression of E-selectin by HUVECs, and the addition of anti-TNF-alpha antibodies significantly reduced that activation. These results are consistent with our hypothesis that intermittent perfusion of the placenta, secondary to reduced trophoblast invasion, causes increased secretion of TNF-alpha, and that this contributes to the activation of maternal endothelial cells that characterizes preeclampsia.

VEGF mRNA levels in placentae from pregnancies complicated by pre‐eclampsia

Objective To measure the mRNA levels of vascular endothelial growth factor and its receptor in the placenta following delivery after uncomplicated pregnancy and after pregnancies complicated by pre‐eclampsia.

The Effect of Delaying Childbirth on Primary Cesarean Section Rates

Background The relationship between population trends in delaying childbirth and rising rates of primary cesarean delivery is unclear. The aims of the present study were (1) to characterize the association between maternal age and the outcome of labor, (2) to determine the proportion of the increase in primary cesarean rates that could be attributed to changes in maternal age distribution, and (3) to determine whether the contractility of uterine smooth muscle (myometrium) varied with maternal age. Methods and Findings We utilized nationally collected data from Scotland, from 1980 to 2005, and modeled the risk of emergency cesarean section among women delivering a liveborn infant in a cephalic presentation at term. We also studied isolated myometrial strips obtained from 62 women attending for planned cesarean delivery in Cambridge, England, from 2005 to 2007. Among 583,843 eligible nulliparous women, there was a linear increase in the log odds of cesarean delivery with advancing maternal age from 16 y upwards, and this increase was unaffected by adjustment for a range of maternal characteristics (adjusted odds ratio for a 5-y increase 1.49, 95% confidence interval [CI] 1.48–1.51). Increasing maternal age was also associated with a longer duration of labor (0.49 h longer for a 5-y increase in age, 95% CI 0.46–0.51) and an increased risk of operative vaginal birth (adjusted odds ratio for a 5-y increase 1.49, 95% CI 1.48–1.50). Over the period from 1980 to 2005, the cesarean delivery rate among nulliparous women more than doubled and the proportion of women aged 30–34 y increased 3-fold, the proportion aged 35–39 y increased 7-fold, and the proportion aged ≥40 y increased 10-fold. Modeling indicated that if the age distribution had stayed the same over the period of study, 38% of the additional cesarean deliveries would have been avoided. Similar associations were observed in multiparous women. When studied in vitro, increasing maternal age was associated with reduced spontaneous activity and increased likelihood of multiphasic spontaneous myometrial contractions. Conclusions Delaying childbirth has significantly contributed to rising rates of intrapartum primary cesarean delivery. The association between increasing maternal age and the risk of intrapartum cesarean delivery is likely to have a biological basis.

Endoplasmic reticulum stress exacerbates ischemia-reperfusion-induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells.

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 down‐regulation 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 reoxy‐genation‐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 ischemiareperfusion‐induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells. FASEB J. 21, 872–884 (2007)

Endometrial-Peritoneal Interactions during Endometriotic Lesion Establishment

The pathophysiology of endometriosis remains unclear but involves a complex interaction between ectopic endometrium and host peritoneal tissues. We hypothesized that disruption of this interaction would suppress endometriotic lesion formation. We hoped to delineate the molecular and cellular dialogue between ectopic human endometrium and peritoneal tissues in nude mice as a first step toward testing this hypothesis. Human endometrium was xenografted into nude mice, and the resulting lesions were analyzed using microarrays. A novel technique was developed that unambiguously determined whether RNA transcripts identified via microarray analyses originated from human cells (endometrium) or mouse cells (mesothelium). Four key pathways (ubiquitin/proteasome, inflammation, tissue remodeling/repair, and ras-mediated oncogenesis) were revealed, demonstrating communication between host mesothelial cells and ectopic endometrium. Morphometric analysis of nude mouse lesions confirmed that necrosis, inflammation, healing and repair, and cell proliferation occurred during xenograft development. These processes were entirely consistent with the molecular networks revealed by the microarray data. The transcripts detected in the xenografts overlapped with differentially expressed transcripts in a comparison between paired eutopic and ectopic endometria from human endometriotic patients. For the first time, components of the interaction between ectopic endometrium and peritoneal stromal tissues are revealed. Targeted disruption of this dialogue is likely to inhibit endometriotic tissue formation and may prove to be an effective therapeutic strategy for endometriosis.

Independent component analysis of microarray data in the study of endometrial cancer

Gene microarray technology is highly effective in screening for differential gene expression and has hence become a popular tool in the molecular investigation of cancer. When applied to tumours, molecular characteristics may be correlated with clinical features such as response to chemotherapy. Exploitation of the huge amount of data generated by microarrays is difficult, however, and constitutes a major challenge in the advancement of this methodology. Independent component analysis (ICA), a modern statistical method, allows us to better understand data in such complex and noisy measurement environments. The technique has the potential to significantly increase the quality of the resulting data and improve the biological validity of subsequent analysis. We performed microarray experiments on 31 postmenopausal endometrial biopsies, comprising 11 benign and 20 malignant samples. We compared ICA to the established methods of principal component analysis (PCA), Cyber-T, and SAM. We show that ICA generated patterns that clearly characterized the malignant samples studied, in contrast to PCA. Moreover, ICA improved the biological validity of the genes identified as differentially expressed in endometrial carcinoma, compared to those found by Cyber-T and SAM. In particular, several genes involved in lipid metabolism that are differentially expressed in endometrial carcinoma were only found using this method. This report highlights the potential of ICA in the analysis of microarray data.

Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.

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.