Mitochondrial - Endoplasmic reticulum interactions in the trophoblast: Stress and senescence.

Placental stress has been implicated in the pathophysiology of complications of pregnancy, including growth restriction and pre-eclampsia. Initially, attention focused on oxidative stress, but recently mitochondrial and endoplasmic reticulum stress have been identified. Complex molecular interactions exist among these different forms of stress, making it unlikely that any occurs in isolation. In part, this is due to close physiological connections between the two organelles principally involved, mitochondria and the endoplasmic reticulum (ER), mediated through Ca2+ signalling. Here, we review the involvement of the mitochondria-ER unit in the generation of stress within the trophoblast, and consider consequences for obstetric outcome. Mild stress may induce adaptive responses, including upregulation of antioxidant defences and autophagy, while moderate levels may affect stem cell behaviour and reduce cell proliferation, contributing to the growth-restricted phenotype. High levels of stress can stimulate release of pro-inflammatory cytokines and anti-angiogenic factors, increasing the risk of pre-eclampsia. In addition, chronic stress may promote senescence of the trophoblast, which in other cell types leads to a pro-inflammatory senescence-associated secretory phenotype. Evidence from rodents suggests that a degree of trophoblastic stress develops with increasing gestational age in normal pregnancies. The increase in maternal concentrations of soluble fms-like tyrosine kinase-1 (sFlt-1) and reduction in placental growth factor (PlGF) suggest the same may occur in the human, starting around 30 weeks of pregnancy. Placental malperfusion, or co-existing maternal conditions, such as diabetes, will exacerbate that stress. Amelioration of trophoblastic stress should remain a research priority, but will be difficult due to the complexity of the molecular pathways involved.The authors thank all their colleagues and collaborators who have contributed to their studies cited here. GJB gratefully acknowledges the support of the Wellcome Trust (069027/Z/02/Z and 084804/2/08/Z), the MRC and Tommy’s; AJM thanks the Research Councils UK, Action Medical Research (SP4545) and the WYNG Foundation of Hong Kong.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.placenta.2016.04.00

Endoplasmic reticulum stress is induced in the human placenta during labour.

Placental endoplasmic reticulum (ER) stress has been postulated in the pathophysiology of pre-eclampsia (PE) and intrauterine growth restriction (IUGR), but its activation remains elusive. Oxidative stress induced by ischaemia/hypoxia-reoxygenation activates ER stress in vitro. Here, we explored whether exposure to labour represents an in vivo model for the study of acute placental ER stress. ER stress markers, GRP78, P-eIF2α and XBP-1, were significantly higher in laboured placentas than in Caesarean-delivered controls localised mainly in the syncytiotrophoblast. The similarities to changes observed in PE/IUGR placentas suggest exposure to labour can be used to investigate induction of ER stress in pathological placentas.This study was funded by the Wellcome Trust (Grant No. 084804/2/08/Z) in UK and Ter Meulen Fund, Royal Netherlands Academy of Arts and Sciences in Netherlands.This is the final published version. It originally appeared online at http://www.sciencedirect.com/science/article/pii/S0143400414008340#

Morphological and molecular changes in the murine placenta exposed to normobaric hypoxia throughout pregnancy.

Chronic hypoxia is a common complication of pregnancy, arising through malperfusion of the placenta or pregnancy at high altitude. The present study investigated the effects of hypoxia on the growth of the placenta, which is the organ that interfaces between the mother and her fetus. Mice were housed in an hypoxic environment for the whole of gestation. An atmosphere of 13% oxygen induced fetal growth restriction (1182 ± 9 mg, n = 90 vs. 1044 ± 11 mg, n = 62, P < 0.05) but enhanced placental weight (907 ± 11 mg, n = 90 vs. 998 ± 15 mg, n = 62,P < 0.05). Stereological analyses revealed an increase in the volume of maternal blood spaces in the placenta, consistent with increased flow. At the molecular level, we observed activation of the protein kinase B (Akt)-mechanistic target of rapamycin growth and proliferation pathway. Chronic hypoxia also triggered mild endoplasmic reticulum stress, a conserved homeostatic response that mediates translational arrest through phosphorylation of eukaryotic initiation factor 2 subunit α. Surprisingly, although subunits of the mitochondrial electron transport chain complexes were reduced at the protein level, there was no evidence of intracellular energy depletion. Finally, we demonstrated sex-specific placental responses to chronic hypoxia. Placentas from male fetuses were heavier (1082 ± 2 mg, n = 30 vs. 928 ± 2 mg, n = 34, P < 0.05) and less susceptible to hypoxia-induced oxidative stress than those from females. Their capacity to adapt may explain why male fetuses were significantly less growth restricted at embryonic day 18.5 than their female counterparts. These findings are consistent with the concept that male fetuses are more aggressive with respect to their nutrient demands, which may place them at greater risk of adverse outcomes under limiting conditions.This study was supported by a grant from the Wellcome Trust (084804/2/08/Z).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1113/JP27107

Influence of speed of sample processing on placental energetics and signalling pathways: implications for tissue collection.

INTRODUCTION: The placenta is metabolically highly active due to extensive endocrine and active transport functions. Hence, placental tissues soon become ischaemic after separation from the maternal blood supply. Ischaemia rapidly depletes intracellular ATP, and leads to activation of stress-response pathways aimed at reducing metabolic demands and conserving energy resources for vital functions. Therefore, this study aimed to elucidate the effects of ischaemia ex vivo as may occur during tissue collection on phosphorylation of placental proteins and kinases involved in growth and cell survival, and on mitochondrial complexes. METHODS: Eight term placentas obtained from normotensive non-laboured elective caesarean sections were kept at room-temperature and sampled at 10, 20, 30 and 45 min after delivery. Samples were analyzed by Western blotting. RESULTS: Between 10 and 45 min the survival signalling pathway intermediates, P-AKT, P-GSK3α and β, P-4E-BP1 and P-p70S6K were reduced by 30-65%. Stress signalling intermediates, P-eIF2α increased almost 3 fold after 45 min. However, other endoplasmic reticulum stress markers and the Heat Shock Proteins, HSP27, HSP70 and HSP90, did not change. Phosphorylation of AMPK, an energy sensor, was elevated 2 fold after 45 min. Contemporaneously, there was an ∼25% reduction in mitochondrial complex IV subunit I. DISCUSSION AND CONCLUSIONS: These results suggest that for placental signalling studies, samples should be taken and processed within 10 min of caesarean delivery to minimize the impact of ischaemia on protein phosphorylation

AMPK and uterine artery vasodilation

Genes near adenosine monophosphate-activated protein kinase-α1 (PRKAA1) have been implicated in the greater uterine artery (UtA) blood flow and relative protection from fetal growth restriction seen in altitude-adapted Andean populations. Adenosine monophosphate-activated protein kinase (AMPK) activation vasodilates multiple vessels but whether AMPK is present in UtA or placental tissue and influences UtA vasoreactivity during normal or hypoxic pregnancy remains unknown. We studied isolated UtA and placenta from near-term C57BL/6J mice housed in normoxia (n = 8) or hypoxia (10% oxygen, n = 7-9) from day 14 to day 19, and placentas from non-labouring sea level (n = 3) or 3100 m (n = 3) women. Hypoxia increased AMPK immunostaining in near-term murine UtA and placental tissue. RT-PCR products for AMPK-α1 and -α2 isoforms and liver kinase B1 (LKB1; the upstream kinase activating AMPK) were present in murine and human placenta, and hypoxia increased LKB1 and AMPK-α1 and -α2 expression in the high- compared with low-altitude human placentas. Pharmacological AMPK activation by A769662 caused phenylephrine pre-constricted UtA from normoxic or hypoxic pregnant mice to dilate and this dilatation was partially reversed by the NOS inhibitor l-NAME. Hypoxic pregnancy sufficient to restrict fetal growth markedly augmented the UtA vasodilator effect of AMPK activation in opposition to PE constriction as the result of both NO-dependent and NO-independent mechanisms. We conclude that AMPK is activated during hypoxic pregnancy and that AMPK activation vasodilates the UtA, especially in hypoxic pregnancy. AMPK activation may be playing an adaptive role by limiting cellular energy depletion and helping to maintain utero-placental blood flow in hypoxic pregnancy.Funding for these studies was provided by the Wellcome Trust (084804/2/08/Z) to G.J.B., the British Heart Foundation and the Wellcome Trust to D.A.G., the Biotechnology and Biological Sciences Research Council (BBSRC) to A.L.F., a UK Wellcome Trust Programme Grant (WT081195MA) to A.M.E. and A.D.M., a BBSRC studentship and in vivo skills award to J.S.H., a National Health Medical Research Council and Centre for Trophoblast Research fellowship to A.N.S.-P., and a NIH RO1 grant (HLBI-079647) to L.G.M. along with sabbatical support from Wake Forest University.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1113/JP27099

Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B

$\textbf{STUDY QUESTION}$: Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts? $\textbf{SUMMARY ANSWER}$: ET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion. $\textbf{WHAT IS KNOWN ALREADY}$: MMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE. $\textbf{STUDY DESIGN, SIZE, DURATION}$: $\textit{In vitro}$ study using primary human trophoblasts from 50 first trimester placentas (gestational week 7-12). $\textbf{PARTICIPANTS/MATERIALS, SETTING, METHODS}$: Trophoblasts were cultured in the absence or presence of 10-100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting. $\textbf{MAIN RESULTS AND THE ROLE OF CHANCE}$: ET-1 down-regulated MMP14 and MMP15 mRNA (-21% and -26%, respectively, $\textit{P}$ < 0.05) and protein levels (-18% and -22%, respectively, $\textit{P}$ < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (-24%, $\textit{P}$ < 0.05) and trophoblast invasion (-26%, $\textit{P}$ ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, $\textit{P}$ < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs. $\textbf{LARGE SCALE DATA}$: N/A. $\textbf{LIMITATIONS, REASONS FOR CAUTION}$: Only primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5-8% O2) were not analyzed in this study. $\textbf{WIDER IMPLICATIONS OF THE FINDINGS}$: ET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE.The study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfelder'sche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN)

Exposure to fluctuating salinity enhances free amino acid accumulation in Tigriopus californicus (Copepoda)

Intracellular concentrations of free amino acids (FAA) in the intertidal copepod Tigriopus californicus increase in response to hyperosmotic stress and decrease in response to hypo-osmotic stress. The purpose of this study was to determine if exposure to repeated bouts of osmotic stress resulted in changes in FAA accumulation or the degree of FAA retention in subsequent episodes. Five groups of T. californicus were exposed for 22 days to a fluctuating salinity regime which consisted of 24 h at 100% seawater followed by 24 h at either 90, 80, 70, 60 or 50% seawater (11 cycles). After the tenth exposure to 100% seawater, individuals from each treatment group were analyzed for alanine and proline concentration. Alanine and proline accumulation generally increased in proportion to the osmotic stress up to 60–100% seawater — additional osmotic stress failed to increase total accumulation. Prior exposure to fluctuating salinity increased the extent of alanine and proline retention observed upon transfer to a hypo-osmotic medium. The treatment group which had experienced the most extreme fluctuation (50–100% seawater) retained alanine and proline levels approximately 10- and 20-fold higher, respectively, than controls. A less severe salinity fluctuation was required to elicit this response for alanine (90–100% seawater) than for proline (60–100% seawater). Previous exposure to fluctuating salinity also resulted in increased alanine and proline accumulation in subsequent episodes of hyperosmotic stress. 24 h after transfer from 50 to 100% seawater, alanine and proline levels in the conditioned copepods were approximately 3- and 7-fold higher, respectively, than in copepods which had not been cycled. This facilitation in alanine and proline accumulation occurred after 10 and 11 cycles, respectively. Of the increased accumulation in alanine and proline, 7.0% and 22.5%, respectively, could be accounted for by the higher degree of FAA retention while under hypo-osmotic conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47130/1/360_2004_Article_BF00692733.pd

Confidence Arguments for Evidence of Performance in Machine Learning for Highly Automated Driving Functions

Due to their ability to efficiently process unstructured and highly dimensional input data, machine learning algorithms are being applied to perception tasks for highly automated driving functions. The consequences of failures and insu_ciencies in such algorithms are severe and a convincing assurance case that the algorithms meet certain safety requirements is therefore required. However, the task of demonstrating the performance of such algorithms is non-trivial, and as yet, no consensus has formed regarding an appropriate set of verification measures. This paper provides a framework for reasoning about the contribution of performance evidence to the assurance case for machine learning in an automated driving context and applies the evaluation criteria to a pedestrian recognition case study