Placental expression of adenosine A(2A) receptor and hypoxia inducible factor-1 alpha in early pregnancy, term and pre-eclamptic pregnancies: interactions with placental renin-angiotensin system.

Hypoxia-inducible factors (HIFs), adenosine and tissue renin-angiotensin-system (RAS) promote angiogenesis and vascularisation. We investigated the temporal expression placental adenosine A2AR receptor and HIF-1α in early pregnancy and at delivery in normotensive (NT) and pre-eclamptic (PE) women. Results were compared to our previously reported angiotensin receptor data. Expression of A2AR and HIF-1α was highest at ≤10 weeks, positively correlated through pregnancy and was higher in PE than NT at delivery. The A2AR associated with the AT4R only in early pregnancy. We suggest adenosine and RAS may interact to promote placentation with a potential adaptation to poor placental perfusion in PE.

Placental expression of eNOS, iNOS and the major protein components of caveolae in women with pre-eclampsia.

Caveolae regulate many cardiovascular functions and thus could be of interest in relation to pre-eclampsia, a pregnancy specific disorder characterised by hypertension and proteinuria. We examined placental mRNA and protein expression/localisation of the caveolae components Caveolin 1-3, Cavin 1-4 as well as eNOS/iNOS in normotensive control (n = 24) and pre-eclamptic pregnancies (n = 19). Placental mRNA expression of caveolin-1, cavin 1-3, was lower and eNOS expression was increased in pre-eclampsia (P < 0.05 for all). Additionally Caveolin-1 protein expression was also reduced in pre-eclampsia (P = 0.007); this could be an adaptive response in pre-eclampsia, possibly to attenuate the oxidative stress/inflammation.

Expression of voltage-dependent potassium channels in first trimester human placentae.

Potassium channel α-subunits encoded by KCNQ1-5 genes form voltage-dependent channels (KV7), modulated by KCNE1-5 encoded accessory proteins. The aim was to determine KCNQ and KCNE mRNA expression and assess protein expression/localisation of the KCNQ3 and KCNE5 isoforms in first trimester placental tissue. Placentae were obtained from women undergoing elective surgical termination of pregnancy (TOP) at ≤ 10 weeks' (early TOP) and >10 weeks' (mid TOP) gestations. KCNQ1-5 expression was unchanged during the first trimester. KCNE5 expression increased in mid TOP vs. early TOP samples (P = 0.022). This novel study reports mRNA and protein expression of KV7 channels in first trimester placentae.

Thyroid hormones and their placental deiodination in normal and pre-eclamptic pregnancy.

Pre-eclampsia is associated with lower serum selenium concentrations and glutathione peroxidase expression/activity; total thyroid hormones are also lower. OBJECTIVES, STUDY DESIGN AND MAIN OUTCOME MEASURES: We hypothesised that the placental selenoprotein deiodinase (D3) will be protected in pre-eclampsia due to the hierarchy of selenoprotein biosynthesis in selenium deficiency. Venous blood and tissue from three standardised placental sites were obtained at delivery from 27 normotensive and 23 pre-eclamptic women. mRNA expression and enzyme activity were assessed for both deiodinases (D2 and D3); protein expression/localisation was also measured for D3. FT4, FT3 and TSH concentrations were measured in maternal and umbilical cord blood. RESULTS: No significant differences in D3 mRNA or protein expression between normotensive and pre-eclamptic pregnancies. There was a significant effect of sampling site on placental D3 activity only in pre-eclamptic women (P = 0.034; highest activity nearest the cord). A strong correlation between D3 mRNA expression and enzyme activity existed only in the pre-eclamptic group; further strengthened when controlling for maternal selenium (P < 0.002). No significant differences were observed between groups for any of the maternal thyroid hormones; umbilical TSH concentrations were significantly higher in the pre-eclamptic samples (P < 0.001). CONCLUSIONS: D3 mRNA and protein expression appear to be independent of selenium status. Nevertheless, the positive correlation between D3 mRNA expression and activity evident only in pre-eclampsia, suggests that in normotensive controls, where selenium is higher, translation is not affected, but in pre-eclampsia, where selenium is low, enzyme regulation may be altered. The raised umbilical TSH concentrations in pre-eclampsia may be an adaptive fetal response to maximise iodide uptake.

The placental renin-angiotensin system and oxidative stress in pre-eclampsia.

There is an inverse correlation between human birthweight and umbilical venous angiotensin II (AngII) concentrations. Oxidative stress and increased pro-renin receptor (PRR) both enhance the cleavage of angiotensin I from angiotensinogen (AGT). Pre-eclampsia, a hypertensive disorder of pregnancy, manifests as high blood pressure and proteinuria, and is a state of increased oxidative stress. HYPOTHESIS: Pre-eclampsia will be associated with increased placental expression of components of the renin-angiotensin system, which could result in reduced infant birthweight. Biopsies were taken 1 cm from the placental edge from 27 normotensive controls and 23 pre-eclamptic White European women. Immunohistochemistry was performed for AGT, PRR, glutathione peroxidase 3 (GPx3) and the AT1R and AT2R AngII receptors. Protein expression was semi-quantitatively assessed (H-score). RESULTS: AT1R expression was significantly increased in pre-eclamptic placentae, and negatively correlated with birthweight (r = -0.529, P = 0.009). AT1R expression was also negatively correlated with GPx3 expression overall (r = -0.647; P = 0.005). AT2R expression positively correlated with AGT (r = 0.615, P = 0.002) in the pre-eclamptic placentae only. CONCLUSIONS: The raised AT1R expression in pre-eclampsia, together with inadequate antioxidant protection, possibly through lower GPx activity, might enhance the vasoconstrictor effect of locally-generated AngII, contributing to the restricted fetal growth characteristic of pre-eclampsia. Conversely, the AT2R:AGT association within the pre-eclamptic placenta may provide a compensatory mechanism.

OS081. Novel KCNQ3/KCNE5 isoform protein and mRNA expression in first trimester human placentae.

INTRODUCTION: Potassium channel α-subunits encoded by KCNQ1-5 genes (Kv7) form voltage-dependent channels that can be modulated by KCNE1-5 encoded accessory proteins. These channels are known to play a role in the reactivity of blood vessels. We have previously shown that both mRNA and protein expression for the novel combination of KCNQ3 and KCNE5 are increased in term and preterm pre-eclampsia (PE) compared to normotensive control placentae [1]. The expression of these isoforms in early placental tissue has not been examined. OBJECTIVES: The aims of this study were to determine whether KCNQ3 and KCNE5 mRNA and proteins are expressed in first trimester placental tissue. METHODS: Placental samples were obtained from women undergoing elective surgical termination of pregnancy between 6 and 12 weeks' gestation (n=7) following informed written consent. KCNQ3 and KCNE5 mRNA expression was measured by qRT-PCR and normalised to stably expressed GAPDH. Immunohistochemistry was used to assess protein expression and localisation of the isoforms. RESULTS: Both mRNA and protein expression of KCNQ3 and KCNE5 were detected in placental tissue at all gestations. KCNE5 mRNA expression remained constant between 6 and 10 weeks with a subsequent rise at 11 and 12 weeks. KCNQ3 mRNA expression was initially lower than KCNE5 but markedly increased at 7 weeks remaining high until 10 weeks and falling below KCNE5 levels by 12 weeks. Protein expression for both KCNQ3 and KCNE5 was localised mainly to the syncytiotrophoblast but was also evident in the mesenchyme; overall KCNQ3 intensity significantly increased with gestational age (p=0.044). CONCLUSION: KCNQ3 and KCNE5 channel isoforms are highly expressed in first trimester placenta. The temporal changes in mRNA expression mirror changes in the placental tissue oxygen tension which increases between 8 and 10 weeks. This would precede the dislocation of the spiral artery plugs enabling maternal blood to flow freely and continuously into the intervillous spaces. We speculate that the increase in mesenchymal protein expression may be related to angiogenesis during this critical window of feto-placental vascular development. Future work will characterise the complete KCNQ/KCNE isoforms in first trimester placental tissue and assess potential functional roles of these channels both in early placentation and in relation to PE. FUNDING: Tommy's Charity (Registered charity 1060508).

Expression of AT1R, AT2R and AT4R and their roles in extravillous trophoblast invasion in the human.

The placental renin-angiotensin system (RAS) is active from early pregnancy and may have a role in placentation. Angiotensin II (AngII) acts via binding to receptor types AT1R and AT2R. Recently smaller peptide members of the angiotensin family have been recognised as having biological relevance. Angiotensin (3-8) (AngIV) has a specific receptor (AT4R) and evokes hypertrophy, vasodilatation and vascular inflammatory response. The aim of this study was to characterise placental expression of AT1R, AT2R and AT4R, and to determine whether AngII and AngIV regulate extravillous trophoblast (EVT) invasion, apoptosis and proliferation. Placental samples were obtained from women undergoing elective surgical termination of pregnancy (TOP) at 8-10 weeks gestation (early TOP), 12-14 weeks gestation (mid TOP) or at delivery following normal pregnancy or with pre-eclampsia (PE). Immunohistochemistry and qRT-PCR were performed to determine placental mRNA and protein expression of AT1R, AT2R and AT4R at all gestational ages. EVT invasion following culture with AngII or AngIV was assessed in early placental tissue using Matrigel invasion assays. Invasion was assessed on day 6 of culture and placental explants were harvested for immunohistochemical analysis of apoptosis and proliferation. The results from qRT-PCR and immunohistochemistry showed placental AT1R expression which did not vary with gestation. The highest levels of expression of AT2R were found in early and mid TOP placentae compared to term pregnancy. Expression of AT4R was increased in term placentae, with a significant reduction in PE placentae. Moreover, culture with AngIV or AngII increased EVT invasion from placental explants, which showed increased trophoblast proliferation and reduced apoptosis. This study has characterised expression of AT4R and AT1R and AT2R in human placenta throughout normal pregnancy and in PE. Both AngIV and AngII may play an important role in normal pregnancy.

Differential expression and distribution of placental glutathione peroxidases 1, 3 and 4 in normal and preeclamptic pregnancy.

Preeclampsia is a pregnancy-specific condition affecting 2-7% of women and a leading cause of perinatal and maternal morbidity and mortality; it may also predispose the mother and fetus to increased risks of adult cardiovascular disease. The selenoprotein glutathione peroxidases (GPxs) have critical roles in regulating antioxidant status. OBJECTIVES, STUDY DESIGN AND MAIN OUTCOME MEASURES: Immunohistochemical measurements of GPx1, GPx3 and GPx4 protein expression were performed on samples taken from three standardised sampling sites between the cord insertion and the periphery of the placenta from 12 normotensive, and 12 preeclamptic women to establish if their expression differed between sampling sites. Total GPx activities were also examined from the three sampling sites of these placentae. RESULTS: There were highly significant reductions in overall immunohistochemical staining of all 3 GPxs in the preeclampsia compared to normotensive placentae (GPx1: P=0.016; GPx3: P=0.003; GPx4: P<0.001). Furthermore, graded differences in expression between the standardised placental sampling sites were also found for GPx3 (higher in the inner region, P=0.05) and GPx4 (higher in the periphery, P=0.02) but not GPx1. Placental GPx enzyme activity was also significantly reduced in tissue from preeclamptic women as compared to normotensive women (P=0.007; the difference was more pronounced nearest the cord insertion). CONCLUSIONS: We have shown highly significant reductions in expression of all three major classes of GPx in placentae from women with preeclampsia, and distribution gradients in activity, which may relate to the differential oxygenation of regions of the placenta.

The non-invasive biopsy--will urinary proteomics make the renal tissue biopsy redundant?

Proteomics is a rapidly advancing technique which gives functional insight into gene expression in living organisms. Urine is an ideal medium for study as it is readily available, easily obtained and less complex than other bodily fluids. Considerable progress has been made over the last 5 years in the study of urinary proteomics as a diagnostic tool for renal disease. Advantages over the traditional renal biopsy include accessibility, safety, the possibility of serial sampling and the potential for non-invasive prognostic and diagnostic monitoring of disease and an individual's response to treatment. Urinary proteomics is now moving from a discovery phase in small studies to a validation phase in much larger numbers of patients with renal disease. Whilst there are still some limitations in methodology, which are assessed in this review, the possibility of urinary proteomics replacing the invasive tissue biopsy for diagnosis of renal disease is becoming an increasingly realistic option.