Clare Whitehead

Peroxisome proliferator-activated receptors are altered in pathologies of the human placenta: Gestational diabetes mellitus, intrauterine growth restriction and preeclampsia

BACKGROUND Common complications of pregnancy arise in part from dysfunctional placental development, and include gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia (PE). Peroxisome proliferator-activated receptors (PPARs), and their partner retinoid X receptor a (RXRalpha), mediate trophoblast differentiation and thus may offer insight into the pathophysiology of these diseases. METHODS Human placentae were obtained from women at term with GDM and were compared to uncomplicated term placentae. Placentae from women who delivered preterm with IUGR, PE or co-existing PE and IUGR were compared to matched controls. Quantitative RT-PCR and Western blotting were used to examine mRNA and protein expression of PPARalpha, PPARdelta, PPARgamma and RXRalpha. DNA binding activity of PPAR isoforms were measured in nuclear protein extracts. RESULTS GDM was associated with significantly lower placental PPARgamma mRNA and protein, PPARalpha protein and RXRalpha protein expression, while PPAR DNA binding activity remained unchanged. Placentae from women with PE did not demonstrate any changes in mRNA or protein expression or PPAR DNA binding activity, while IUGR/PE placenta showed significant increases in PPARalpha protein, PPARgamma mRNA and protein and RXRalpha mRNA and protein expression. Significantly elevated protein expression of PPARalpha and RXRalpha were associated with IUGR placentae. IUGR and IUGR/PE placentae had significantly higher PPARgamma DNA binding activity compared to controls. CONCLUSIONS The data presented herein suggest that PPARs may be involved in the pathophysiology of GDM, PE and IUGR.

MMP-14 Is Expressed in Preeclamptic Placentas and Mediates Release of Soluble Endoglin

Soluble endoglin is an anti-angiogenic protein that is released from the placenta and contributes to both maternal endothelial dysfunction and the clinical features of severe preeclampsia. The mechanism through which soluble endoglin is released from the placenta is currently unknown; however, recent work in colorectal cancer identified matrix metalloproteinase 14 (MMP-14) as the cleavage protease of endoglin. To determine whether this is also the mechanism responsible for soluble endoglin release in preeclampsia, we investigated the expression of MMP-14 within the placenta and the effects of its inhibition on soluble endoglin release. Placentas were obtained from severe, early onset preeclamptic pregnancies (n = 8) and gestationally matched preterm controls (n = 8). MMP-14 was predominately localized to the syncytiotrophoblast. Results from a proximity ligation assay showed protein interactions between endogenous MMP-14 and endoglin within the preeclamptic placenta. To demonstrate that this interaction produces soluble endoglin, we treated trophoblastic BeWo cells with either a broad-spectrum MMP inhibitor (GM6001) or MMP-14 siRNA. Both treatments produced a decrease in soluble endoglin (P ≤ 0.05). Treatment of mice bearing BeWo xenografts with GM6001 decreased circulating soluble endoglin levels in mouse serum (P ≤ 0.05). These findings indicate that MMP-14 is the likely cleavage protease of endoglin in the setting of preeclampsia. This approach provides a novel method for the development of potential therapeutics to reduce circulating soluble endoglin and ameliorate the clinical features of severe preeclampsia.

Circulating MicroRNAs in Maternal Blood as Potential Biomarkers for Fetal Hypoxia In-Utero

Stillbirth affects 1 in 200 pregnancies and commonly arises due to a lack of oxygen supply to the fetus. Current tests to detect fetal hypoxia in-utero lack the sensitivity to identify many babies at risk. Emerging evidence suggests that microRNAs derived from the placenta circulate in the maternal blood during pregnancy and may serve as non-invasive biomarkers for pregnancy complications. In this study, we examined the expression of miRs known to be regulated by hypoxia in two clinical settings of significant fetal hypoxia: 1) labour and 2) fetal growth restriction. Six miRs (miR 210, miR 21, miR 424, miR 199a, miR 20b, and miR 373) were differentially expressed in pregnancies complicated by fetal hypoxia. In healthy term pregnancies there was a 4.2 fold increase in miR 210 (p<0.01), 2.7 fold increase in miR 424 (p<0.05), 2.6 fold increase in miR 199a (p<0.01) and 2.3 fold increase in miR 20b (p<0.05) from prior to labour to delivery of the fetus. Furthermore, the combined expression of miR 21 and miR 20b correlated with the degree of fetal hypoxia at birth determined by umbilical cord lactate delivery (r = 0.79, p = 0.03). In pregnancies complicated by severe preterm fetal growth restriction there was upregulation of the hypoxia-regulated miRs compared to gestation-matched controls: 3.6 fold in miR 210 (p<0.01), 3.6 fold in miR 424 (p<0.05), 5.9 fold in miR 21 (p<0.01), 3.8 fold in miR 199a (p<0.01) and 3.7 fold in miR 20b (p<0.01). Interestingly, the expression of miR 373 in gestation matched controls was very low, but was very highly expressed in FGR (p<0.0001). Furthermore, the expression increased in keeping with the degree of in-utero hypoxia estimated by fetal Doppler velocimetry. We conclude quantifying hypoxia-regulated miRs in the maternal blood may identify pregnancies at risk of fetal hypoxia, enabling early intervention to improve perinatal outcomes.

Characterization of symptoms immediately preceding eclampsia.

OBJECTIVE: To characterize the symptoms that immediately precede eclamptic seizures. METHODS: We did a prospective observational study of all women admitted to a single center in Tanzania between May 1, 2007 and April 30, 2008 who had an eclamptic seizure. During their admission they were asked a uniform set of questions related to symptoms preceding the seizure. RESULTS: There were 3,267 deliveries and 46 cases of eclampsia (1.4%). Neurologic symptoms (headache [80%] with or without visual disturbance [45%]) were the most common prodrome symptoms, regardless of degree of hypertension or whether the seizure occurred antepartum or postpartum. Twenty percent of women with eclampsia reported no neurologic symptoms before seizure. CONCLUSION: Neurologic symptoms commonly precede eclampsia. A minority of patients with eclampsia (17%) had no prodromal symptoms before their eclamptic seizure. Premonitory symptoms may provide an early warning of imminent eclampsia. LEVEL OF EVIDENCE: III

Placental specific mRNA in the maternal circulation are globally dysregulated in pregnancies complicated by fetal growth restriction.

CONTEXT Fetal growth restriction (FGR) is a leading cause of perinatal mortality, yet no reliable screening test exists. Placental specific mRNA in the maternal circulation may reflect changes in the placental transcriptome in FGR and could be a novel biomarker for FGR. OBJECTIVE The aim of the study was to identify placental specific RNA detectable in the maternal circulation and examine whether they are differentially expressed in severe preterm FGR. DESIGN In silico screening was used to identify placental specific RNAs. Their expression in cases of severe FGR vs controls was examined in both maternal blood and placenta by microarray, RT-PCR, and in situ hybridization. RESULTS Via in silico analysis, we identified 137 genes very highly expressed in the placenta relative to other tissues. Using microarray, we found that they were detectable in the maternal blood and were globally dysregulated with preterm FGR; 75 genes (55%) had a ≥1.5-fold differential expression compared to controls. Eight genes (ERVWE-1, PSG1, PLAC4, TAC3, PLAC3, CRH, CSH1, and KISS1) were validated by RT-PCR to be significantly increased in both maternal blood and placenta in a larger cohort of severe FGR compared to controls. In situ hybridization confirmed PAPPA2 and ERVWE-1 localized to the syncytiotrophoblast. CONCLUSION There is global differential expression of placental specific mRNA in the maternal blood in pregnancies complicated by severe preterm FGR. Placental specific mRNA in maternal blood may represent a new class of biomarkers for preterm FGR.

Measurement of mRNA Transcripts of Very High Placental Expression in Maternal Blood as Biomarkers of Preeclampsia

CONTEXT mRNA of placental origin in maternal blood shows potential as a clinical biomarker of obstetric diseases such as preeclampsia (PE). We hypothesized that mRNA transcripts very highly expressed in the placenta relative to other tissues will be differentially expressed in PE and be useful as mRNA biomarkers in maternal blood. OBJECTIVE Our objective was to identify a panel of genes highly expressed in the placenta and compare their expression in placenta and maternal whole blood from PE vs. control pregnancies. SETTING Placental tissue and maternal whole blood specimens were obtained from normotensive controls (n = 15) and pregnancies complicated by severe preterm PE (n = 21). INTERVENTION mRNA expression was evaluated by quantitative real-time RT-PCR. RESULTS We identified 20 genes exhibiting highest to fourth highest expression in the placenta relative to all other tissues. All genes were detectable in placenta. Nine of the 20 genes were detectable in maternal whole blood. Four of the nine genes detectable in blood (i.e. PLAC3, PLAC4, CRH, and ERVWE1) were significantly increased in both maternal blood and placenta from PE pregnancies. The remaining five genes detectable in maternal blood were unchanged in both blood and placenta from PE pregnancies. Thus, there was complete correlation of gene expression between maternal blood and placenta. CONCLUSIONS Circulating mRNA coding genes of high placental expression show strong correlation with transcript levels in preeclamptic placenta. Such transcripts may be promising candidates to screen as mRNA biomarkers of PE in maternal whole blood.

Placental expression of a novel primate-specific splice variant of sFlt-1 is upregulated in pregnancies complicated by severe early onset pre-eclampsia.

Please cite this paper as: Whitehead C, Palmer K, Nilsson U, Gao Y, Saglam B, Lappas M, Tong S. Placental expression of a novel primate‐specific splice variant of sFlt‐1 is upregulated in pregnancies complicated by severe early onset pre‐eclampsia. BJOG 2011; DOI: 10.1111/j.1471‐0528.2011.02962.x.

Effects of gefitinib, an epidermal growth factor receptor inhibitor, on human placental cell growth.

OBJECTIVE: Placenta has the highest expression of epidermal growth factor (EGF) receptor of all tissues, a cell signaling pathway promoting survival and growth. Therefore, EGF receptor inhibition could potentially treat ectopic pregnancy. We undertook preclinical studies to examine whether gefitinib (orally available EGF receptor inhibitor) with or without methotrexate inhibits placental cell growth. METHODS: Gefitinib and methotrexate were added to placental cells and their ability inhibit cell growth, block EGF receptor signaling, and induce apoptosis (programmed cell death) was examined. They were also administered to two animal mouse models to examine their effects on placental tissue in vivo. Results: Epidermal growth factor receptor was highly expressed in placental tissue from ectopic pregnancies. Combining gefitinib with methotrexate potently inhibited growth of placental cells, including placental cell lines (JEG3, BeWo cells) and cells isolated from first-trimester placenta. These drugs were additive in blocking EGF receptor signaling and inducing apoptosis. Gefitinib and methotrexate administered together were more potent in decreasing the volume of human placental cells xenografted subcutaneously onto mice compared with either alone. By day 19 after xenografting, mean (±standard error of the mean), xenograft volumes were: 821 (±68) mm3 after gefitinib treatment, 901 (±204) mm3 after methotrexate treatment, and 345 (±137) mm3 after both drugs were given (P<.01 for both comparisons of single therapy compared with combination therapy). Combining these agents doubled rates of fetal resorption in pregnant mice compared with each drug alone. CONCLUSION: Combining gefitinib with methotrexate potently inhibits placental cell growth in vitro and in mouse models. The combination may have potential in treating ectopic pregnancies.

Placental Insufficiency in Fetuses That Slow in Growth but Are Born Appropriate for Gestational Age: A Prospective Longitudinal Study

Objectives To determine whether fetuses that slow in growth but are then born appropriate for gestational age (AGA, birthweight >10th centile) demonstrate ultrasound and clinical evidence of placental insufficiency. Methods Prospective longitudinal study of 48 pregnancies reaching term and a birthweight >10th centile. We estimated fetal weight by ultrasound at 28 and 36 weeks, and recorded birthweight to determine the relative change in customised weight across two timepoints: 28–36 weeks and 28 weeks-birth. The relative change in weight centiles were correlated with fetoplacental Doppler findings performed at 36 weeks. We also examined whether a decline in growth trajectory in fetuses born AGA was associated with operative deliveries performed for suspected intrapartum compromise. Results The middle cerebral artery pulsatility index (MCA-PI) showed a linear association with fetal growth trajectory. Lower MCA-PI readings (reflecting greater diversion of blood supply to the brain) were significantly associated with a decline in fetal growth, both between 28–36 weeks (p = 0.02), and 28 weeks-birth (p = 0.0002). The MCA-PI at 36 weeks was significantly higher among those with a relative weight centile fall <20%, compared to those with a moderate centile fall of 20–30% (mean MCA-PI 1.94 vs 1.61; p<0.05), or severe centile fall of >30% (mean MCA-PI 1.94 vs 1.56; p<0.01). Of 43 who labored, operative delivery for suspected intrapartum fetal compromise was required in 12 cases; 9/18 (50%) cases where growth slowed, and 3/25 (12%) where growth trajectory was maintained (p = 0.01). Conclusions Slowing in growth across the third trimester among fetuses subsequently born AGA was associated with ultrasound and clinical features of placental insufficiency. Such fetuses may represent an under-recognised cohort at increased risk of stillbirth.

Heme Oxygenase-1 Is Not Decreased in Preeclamptic Placenta and Does Not Negatively Regulate Placental Soluble fms-Like Tyrosine Kinase-1 or Soluble Endoglin Secretion

Elevated placental release of the antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG), is central to the pathophysiology of preeclampsia. It is widely accepted that heme oxygenase-1 (HO-1) is decreased in preeclamptic placenta and negatively regulates sFlt-1 and sENG production. We set out to verify these contentions. There was no difference in HO-1 mRNA or protein levels in preterm preeclamptic placentas (n=17) compared with gestationally matched controls (n=27). In silico analysis of microarray studies did not identify decreased placental HO-1 expression in preeclamptic placenta. Silencing HO-1 in primary trophoblasts did not affect sFlt-1 protein secretion after 24 or 48 hours. Silencing nuclear factor (erythroid-derived 2)-like 2 (transcription factor that upregulates HO-1) in trophoblasts also did not affect sFlt-1 secretion. Administering tin protoporphyrin IX dichloride (HO-1 inhibitor) or cobalt protoporphyrin (HO-1 inducer) into placental explants did not affect sFlt-1 or sENG secretion. Silencing HO-1 in 2 types of primary endothelial cells (human umbilical vein endothelial and uterine microvascular endothelial cells) significantly increased sFlt-1 secretion but not sENG secretion. However, HO-1 silencing selectively increased mRNA expression of sFlt-1 i13 (generically expressed sFlt-1 variant) but not of sFlt-1 e15a (sFlt-1 variant mainly expressed in placenta). Furthermore, adding tin protoporphyrin IX dichloride decreased sFlt-1, whereas adding HO-1 inducers (cobalt protoporphyrin, dimethyl fumarate, and rosiglitazone) either had no effect or increased sFlt-1 or sENG secretion (these trends are opposite to what is expected). We conclude that HO-1 expression is not decreased in preeclamptic placenta and HO-1 does not negatively regulate placental sFlt-1 and sENG secretion in placental or endothelial cells.