Edward Johnstone

R1 and R2* changes in the human placenta in response to maternal oxygen challenge

Pregnancy complications such as preeclampsia and fetal growth restriction are sometimes thought to be caused by placental abnormalities associated with reduced oxygenation. Oxygen‐enhanced MRI (R1 contrast) and BOLD MRI (R2* contrast) have the potential to noninvasively investigate this oxygen environment at a range of gestational ages.

Sphingosine-1-phosphate inhibition of placental trophoblast differentiation through a Gi-coupled receptor response

The failure of placental trophoblasts to differentiate properly is thought to play an important role in the cause of pregnancy disorders such as preeclampsia. We looked at the effects of the bioactive lipid sphingosine-1-phosphate (S1P) on the differentiation of primary human cytotrophoblasts (CTs) into syncytiotrophoblasts (STs) in culture. We found that S1P inhibited CT differentiation measured by human chorionic gonadotropin (hCG) secretion and the expression of placental alkaline phosphatase but had no effect on their fusion into multinucleated syncytialized cells. G-protein-linked S1P receptors 1, 2, and 3 were found in CTs by reverse transcriptase-polymerase chain reaction, and receptor 1 was found by Western blot analysis. Disruption of Gi signaling with pertussis toxin reversed the inhibitory effects of S1P. S1P reduced intracellular cAMP, and the addition of 8-bromo-cAMP reversed S1P inhibition of hCG secretion. Therefore, we suggest that S1P inhibits the differentiation of CTs into STs through Gi-coupled S1P receptor interaction(s), leading to the inhibition of adenylate cyclase and reduced production of intracellular cAMP. This is the first reported effect of S1P on placental trophoblast function.

Differential proteomic analysis of highly purified placental cytotrophoblasts in pre-eclampsia demonstrates a state of increased oxidative stress and reduced cytotrophoblast antioxidant defense.

Proteomics were performed using highly (99.99%) purified cytotrophoblasts from six normal and six pre‐eclamptic placentas. Eleven proteins were found which decreased in pre‐eclampsia (actin, glutathione S‐transferase, peroxiredoxin 6, aldose reductase, heat shock protein 60 (Hsp60), two molecular forms of heat shock protein 70 (Hsp70) β‐tubulin, subunit proteasome, ezrin, protein disulfide isomerase, and phosphoglycerate mutase 1). Only one protein, α‐2‐HS‐glycoprotein (fetuin), was found to increase its expression. Western blots of actin, Hsp70, ezrin, and glutatione S‐transferase confirmed decrease in protein expression. Many of the proteins that decreased are consistent with a state of oxidative stress in the pre‐eclamptic placenta and a decreased cytotrophoblast defense against and response to oxidative stress.

Continuous objective recording of fetal heart rate and fetal movements could reliably identify fetal compromise, which could reduce stillbirth rates by facilitating timely management.

Stillbirth currently affects approximately 1 in every 200 pregnancies in the United Kingdom. Fetuses may exhibit signs of compromise as part of a stress response before stillbirth, including reduced fetal movements (RFM) and fetal heart rate (FHR) alterations. At present, and despite widespread use, current fetal monitoring is not associated with a reduction in perinatal mortality rate (PMR) as signs of fetal compromise are not adequately detected. This may be attributed to inaccuracies resulting from manual interpretation of results or subjective assessment of fetal activity. In addition, signs of compromise often occur only hours or days before fetal death, so may be missed by current monitoring methods, which are performed intermittently. A significant consideration is that correct identification of these signs and consequent intervention can result in the delivery of a healthy baby, thus preventing stillbirth. A hypothesis is presented, proposing prompt detection of fetal compromise with the use of 24-hour continuous objective fetal monitoring. With focus placed on obtaining long-term FHR and fetal movement data, prior interest has been found in developing devices for this purpose. However, introduction into clinical practice has not been achieved. Investigation of the hypothesis will begin with the design of a device to record the mentioned parameters, followed by an appropriate validation process. Should development and testing be successful, an eventual comparison in PMR with the use of continuous fetal monitoring vs current monitoring would address the hypothesis. It is suggested that a timely yet reliable indication of fetal wellbeing obtained via long-term monitoring would allow prompt and appropriate obstetric intervention and consequently reduce PMR.

Absence of PO2 change in fetal brain despite PO2 increase in placenta in response to maternal oxygen challenge.

Magnetic resonance imaging allows the noninvasive observation of Po2 changes between air breathing and oxygen breathing through quantification of the magnetic longitudinal relaxation time T1. Changes in Po2 are proportional to changes in the longitudinal relaxation rate ΔR1 (where ΔR1 = 1/T1oxygen − 1/T1air). Knowledge of this response could inform clinical interventions using maternal oxygen administration antenatally to treat fetal growth restriction. We present in vivo measurements of the response of the fetal–placental unit to maternal hyperoxia.

Dysregulated flow-mediated vasodilatation in the human placenta in fetal growth restriction

A correlation was found between in vivo umbilical artery Doppler velocimetry and resistance to fetal‐side flow in the human ex vivo dually perfused placenta, highlighting that the fetoplacental vascular bed is a key site of resistance to umbilico‐placental flow in pregnancy. We discovered high resistance and poor flow‐mediated vasodilatory responses in placentas from pregnancies associated with fetal growth restriction (FGR). Endothelial cells isolated from the FGR placentas and grown in static and flow culture showed a dysregulated phenotype, with biochemical signalling demonstrating a failed compensatory response to high blood‐flow resistance.

Computational modelling of placental amino acid transfer as an integrated system

Placental amino acid transfer is essential for fetal development and its impairment is associated with poor fetal growth. Amino acid transfer is mediated by a broad array of specific plasma membrane transporters with overlapping substrate specificity. However, it is not fully understood how these different transporters work together to mediate net flux across the placenta. Therefore the aim of this study was to develop a new computational model to describe how human placental amino acid transfer functions as an integrated system. Amino acid transfer from mother to fetus requires transport across the two plasma membranes of the placental syncytiotrophoblast, each of which contains a distinct complement of transporter proteins. A compartmental modelling approach was combined with a carrier based modelling framework to represent the kinetics of the individual accumulative, exchange and facilitative classes of transporters on each plasma membrane. The model successfully captured the principal features of transplacental transfer. Modelling results clearly demonstrate how modulating transporter activity and conditions such as phenylketonuria, can increase the transfer of certain groups of amino acids, but that this comes at the cost of decreasing the transfer of others, which has implications for developing clinical treatment options in the placenta and other transporting epithelia.

Placental Features of Late-Onset Adverse Pregnancy Outcome

Objective Currently, no investigations reliably identify placental dysfunction in late pregnancy. To facilitate the development of such investigations we aimed to identify placental features that differ between normal and adverse outcome in late pregnancy in a group of pregnancies with reduced fetal movement. Methods Following third trimester presentation with reduced fetal movement (N = 100), placental structure ex vivo was measured. Placental function was then assessed in terms of (i) chorionic plate artery agonist responses and length-tension characteristics using wire myography and (ii) production and release of placentally derived hormones (by quantitative polymerase chain reaction and enzyme linked immunosorbant assay of villous tissue and explant conditioned culture medium). Results Placentas from pregnancies ending in adverse outcome (N = 23) were ~25% smaller in weight, volume, length, width and disc area (all p<0.0001) compared with those from normal outcome pregnancies. Villous and trophoblast areas were unchanged, but villous vascularity was reduced (median (interquartile range): adverse outcome 10 (10–12) vessels/mm2 vs. normal outcome 13 (12–15), p = 0.002). Adverse outcome pregnancy placental arteries were relatively insensitive to nitric oxide donated by sodium nitroprusside compared to normal outcome pregnancy placental arteries (50% Effective Concentration 30 (19–50) nM vs. 12 (6–24), p = 0.02). Adverse outcome pregnancy placental tissue contained less human chorionic gonadotrophin (20 (11–50) vs. 55 (24–102) mIU/mg, p = 0.007) and human placental lactogen (11 (6–14) vs. 27 (9–50) mg/mg, p = 0.006) and released more soluble fms-like tyrosine kinase-1 (21 (13–29) vs. 5 (2–15) ng/mg, p = 0.01) compared with normal outcome pregnancy placental tissue. Conclusion These data provide a description of the placental phenotype of adverse outcome in late pregnancy. Antenatal tests that accurately reflect elements of this phenotype may improve its prediction.

Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero‐trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanisms mediating [14C]l‐serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that l‐serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [14C]l‐serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero‐trans uptake. Both L‐type amino acid transporter (LAT) 1 and LAT2 subtypes of system L were distributed to MVM, with L‐serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.—Widdows, K. L., Panitchob, N., Crocker, I. P., Please, C. P., Hanson, M. A., Sibley, C. P., Johnstone, E. D., Sengers, B. G., Lewis, R. M., Glazier, J. D. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms. FASEB J. 29, 2583‐2594 (2015). www.fasebj.org

Phenylalanine transfer across the isolated perfused human placenta: an experimental and modeling investigation.

Membrane transporters are considered essential for placental amino acid transfer, but the contribution of other factors, such as blood flow and metabolism, is poorly defined. In this study we combine experimental and modeling approaches to understand the determinants of [14C]phenylalanine transfer across the isolated perfused human placenta. Transfer of [14C]phenylalanine across the isolated perfused human placenta was determined at different maternal and fetal flow rates. Maternal flow rate was set at 10, 14, and 18 ml/min for 1 h each. At each maternal flow rate, fetal flow rates were set at 3, 6, and 9 ml/min for 20 min each. Appearance of [14C]phenylalanine was measured in the maternal and fetal venous exudates. Computational modeling of phenylalanine transfer was undertaken to allow comparison of the experimental data with predicted phenylalanine uptake and transfer under different initial assumptions. Placental uptake (mol/min) of [14C]phenylalanine increased with maternal, but not fetal, flow. Delivery (mol/min) of [14C]phenylalanine to the fetal circulation was not associated with fetal or maternal flow. The absence of a relationship between placental phenylalanine uptake and net flux of phenylalanine to the fetal circulation suggests that factors other than flow or transporter-mediated uptake are important determinants of phenylalanine transfer. These observations could be explained by tight regulation of free amino acid levels within the placenta or properties of the facilitated transporters mediating phenylalanine transport. We suggest that amino acid metabolism, primarily incorporation into protein, is controlling free amino acid levels and, thus, placental transfer.