Elizabeth Cowley

Placental-specific Igf2 knockout mice exhibit hypocalcemia and adaptive changes in placental calcium transport

Evidence is emerging that the ability of the placenta to supply nutrients to the developing fetus adapts according to fetal demand. To examine this adaptation further, we tested the hypothesis that placental maternofetal transport of calcium adapts according to fetal calcium requirements. We used a mouse model of fetal growth restriction, the placental-specific Igf2 knockout (P0) mouse, shown previously to transiently adapt placental System-A amino acid transporter activity relative to fetal growth. Fetal and placental weights in P0 mice were reduced when compared with WT at both embryonic day 17 (E17) and E19. Ionized calcium concentration [Ca2+] was significantly lower in P0 fetal blood compared with both WT and maternal blood at E17 and E19, reflecting a reversal of the fetomaternal [Ca2+] gradient. Fetal calcium content was reduced in P0 mice at E17 but not at E19. Unidirectional maternofetal calcium clearance (Ca K mf) was not different between WT and P0 at E17 but increased in P0 at E19. Expression of the intracellular calcium-binding protein calbindin-D9K, previously shown to be rate-limiting for calcium transport, was increased in P0 relative to WT placentas between E17 and E19. These data show an increased placental transport of calcium from E17 to E19 in P0 compared to WT. We suggest that this is an adaptation in response to the reduced fetal calcium accumulation earlier in gestation and speculate that the ability of the placenta to adapt its supply capacity according to fetal demand may stretch across other essential nutrients.

Sildenafil citrate increases fetal weight in a mouse model of fetal growth restriction with a normal vascular phenotype.

Fetal growth restriction (FGR) is defined as the inability of a fetus to achieve its genetic growth potential and is associated with a significantly increased risk of morbidity and mortality. Clinically, FGR is diagnosed as a fetus falling below the 5th centile of customised growth charts. Sildenafil citrate (SC, Viagra™), a potent and selective phosphodiesterase-5 inhibitor, corrects ex vivo placental vascular dysfunction in FGR, demonstrating potential as a therapy for this condition. However, many FGR cases present without an abnormal vascular phenotype, as assessed by Doppler measures of uterine/umbilical artery blood flow velocity. Thus, we hypothesized that SC would not increase fetal growth in a mouse model of FGR, the placental-specific Igf2 knockout mouse, which has altered placental exchange capacity but normal placental blood flow. Fetal weights were increased (by 8%) in P0 mice following maternal SC treatment (0.4 mg/ml) via drinking water. There was also a trend towards increased placental weight in treated P0 mice (P = 0.056). Additionally, 75% of the P0 fetal weights were below the 5th centile, the criterion used to define human FGR, of the non-treated WT fetal weights; this was reduced to 51% when dams were treated with SC. Umbilical artery and vein blood flow velocity measures confirmed the lack of an abnormal vascular phenotype in the P0 mouse; and were unaffected by SC treatment. 14C-methylaminoisobutyric acid transfer (measured to assess effects on placental nutrient transporter activity) per g placenta was unaffected by SC, versus untreated, though total transfer was increased, commensurate with the trend towards larger placentas in this group. These data suggest that SC may improve fetal growth even in the absence of an abnormal placental blood flow, potentially affording use in multiple sub-populations of individuals presenting with FGR.

Maternal obesity and its effect on placental cell turnover.

Abstract Background: Maternal obesity is a frequent obstetric risk factor, linked with short- and long-term consequences for mother and child, including foetal overgrowth, growth restriction and stillbirth. The mechanisms underlying these pathologies remain unknown but likely involve the placenta. Aims: To study placental cell turnover in relation to maternal body mass index (BMI). Methods: Term placental villous tissue was randomly sampled from 24 pregnancies, with a range of maternal BMI of 19.5–49.6. Immunohistochemistry was performed for human chorionic gonadotropin, Ki67 and M30 and image analysis used to calculate syncytiotrophoblast area and proliferative and apoptotic indices. Results were compared categorically between women of BMI 18.5–24.9 (normal), BMI 30.0–39.9 (obese classes 1and 2) and BMI 40+ (obese class 3) and continuously against BMI; p < 0.05 by the Kruskal–Wallis test or linear regression was considered statistically significant. Results: Increased maternal BMI was associated with categorical (normal versus obese class 3 and obese classes 1 and 2 versus obese class 3, both p < 0.05) and continuous (r2 = 0.24, p = 0.016) reductions in the proliferative index and a continuous reduction (r2 = 0.17, p = 0.047) in the apoptotic index. Discussion: Maternal obesity is associated with a dose-dependent reduction in placental villous proliferation and apoptosis which may increase susceptibility to adverse pregnancy outcomes.

Chorionic plate arterial function is altered in maternal obesity

Objectives To characterise Chorionic Plate Artery (CPA) function in maternal obesity, and investigate whether leptin exposure reproduces the obese CPA phenotype in normal-BMI women. Study design CPA responses to the thromboxane-A2 mimetic U46619 (pre/post leptin incubation), to the nitric oxide donor sodium nitroprusside (SNP) and the occurrence of tone oscillations (pre/post leptin incubation) were assessed in 46 term placentas from women of normal (18.5–24.9) or obese (>30) Body Mass Index (BMI). Outcome measures Area Under the dose response Curve (AUC), maximum response (Vmax), sensitivity (EC50) to U46619 (pre/post leptin) and SNP; average vessel tone, oscillation amplitude and frequency (pre/post leptin). Results U46619 vasoconstriction was similar between BMI categories (p > 0.05), however vasodilatation to SNP was reduced in obesity (AUC p = 0.02, Vmaxp = 0.04) compared to normal-BMI women. Leptin incubation altered responses to U46619 in both normal-BMI (EC50 at 100 ng/ml leptin; p < 0.05) and obese women (AUC at 50 ng/ml; p < 0.05) but vasomotion was unaffected (p > 0.05). Conclusions Maternal obesity is associated with altered placental vascular function which may adversely affect placental oxygen and nutrient transport, placing the fetus at risk. Leptin incubation altered CPA vascular function but did not reproduce the obese phenotype.

Maternal Obesity Impairs Specific Regulatory Pathways in Human Myometrial Arteries

ABSTRACT Obese women (body mass index ≥30 kg/m2) are at greater risk than normal weight women of pregnancy complications associated with maternal and infant morbidity, particularly the development of cardiovascular disease and metabolic disorders in later life; why this occurs is unknown. Nonpregnant, obese individuals exhibit systemic vascular endothelial dysfunction. We tested the hypothesis that obese pregnant women have altered myometrial arterial function compared to pregnant women of normal (18–24 kg/m2) and overweight (25–29 kg/m2) body mass index. Responses to vasoconstrictors, U46619 (thromboxane mimetic) and arginine vasopressin, and vasodilators, bradykinin and the nitric oxide donor sodium nitroprusside, were assessed by wire myography in myometrial arteries from normal weight (n = 18), overweight (n = 18), and obese (n = 20) women with uncomplicated pregnancies. Thromboxane-prostanoid receptor expression was assessed using immunostaining in myometrial arteries of normal weight and obese women. Vasoconstriction and vasodilatation were impaired in myometrial arteries from obese women with otherwise uncomplicated pregnancies. Disparate agonist responses suggest that vascular function in obese women is not globally dysregulated but may be specific to thromboxane and nitric oxide pathways. Because obesity rates are escalating, it is important to identify the mechanisms underlying impaired vascular function and establish why some obese women compensate for vascular dysfunction and some do not. Future studies are needed to determine whether central adiposity results in an altered endocrine milieu that may promote vascular dysfunction by altering the function of perivascular adipose tissue.

Activation of KV7 channels stimulates vasodilatation of human placental chorionic plate arteries.

INTRODUCTION Potassium (K(+)) channels are key regulators of vascular smooth muscle cell (VSMC) excitability. In systemic small arteries, Kv7 channel expression/activity has been noted and a role in vascular tone regulation demonstrated. We aimed to demonstrate functional Kv7 channels in human fetoplacental small arteries. METHODS Human placental chorionic plate arteries (CPAs) were obtained at term. CPA responses to Kv7 channel modulators was determined by wire myography. Presence of Kv7 channel mRNA (encoded by KCNQ1-5) and protein expression were assessed by RT-PCR and immunohistochemistry/immunofluorescence, respectively. RESULTS Kv7 channel blockade with linopirdine increased CPA basal tone and AVP-induced contraction. Pre-contracted CPAs (AVP; 80 mM K(+) depolarization solution) exhibited significant relaxation to flupirtine, retigabine, the acrylamide (S)-1, and (S) BMS-204352, differential activators of Kv7.1 - Kv7.5 channels. All CPAs assessed expressed KCNQ1 and KCNQ3-5 mRNA; KCNQ2 was expressed only in a subset of CPAs. Kv7 protein expression was confirmed in intact CPAs and isolated VSMCs. DISCUSSION Kv7 channels are present and active in fetoplacental vessels, contributing to vascular tone regulation in normal pregnancy. Targeting these channels may represent a therapeutic intervention in pregnancies complicated by increased vascular resistance.

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration

Introduction Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function. Methods A transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH)2D3. QPCR and immunolocalisation were used to demonstrate EVT S1P receptor expression. Results EVTs express S1P receptors 1, 2 and 3. S1P inhibited EVT migration. This effect was abolished in the presence of the specific S1PR2 inhibitor, JTE-013 (p < 0.05 versus S1P alone) whereas treatment with the S1R1/3 inhibitor, FTY720, had no effect. In other cell types S1PR2 is regulated by vitamin D; here we found that treatment with 1,25(OH)2D3 for 48 or 72 h reduces S1PR2 (4-fold; <0.05), but not R1 and R3, expression. Moreover, S1P did not inhibit the migration of cells exposed to 1,25(OH)2D3 (p < 0.05). Discussion This study demonstrates that although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2/Gα12/13 to activate Rho and thereby acts as potent inhibitor of EVT migration. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Our data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.

The atrial natriuretic peptide (ANP) knockout mouse does not exhibit the phenotypic features of pre-eclampsia or demonstrate fetal growth restriction

The ANP knockout mouse is reported to exhibit pregnancy-associated hypertension, proteinuria and impaired placental trophoblast invasion and spiral artery remodeling, key features of pre-eclampsia (PE). We hypothesized that these mice may provide a relevant model of human PE with associated fetal growth restriction (FGR). Here, we investigated pregnancies of ANP wild type (ANP+/+), heterozygous (ANP+/-) and knockout (ANP−/-) mice. Maternal blood pressure did not differ between genotypes (E12.5, E17.5), and fetal weight (E18.5) was unaffected. Placental weight was greater in ANP−/− versus ANP+/+ mice. Therefore, in our hands, the ANP model does not express phenotypic features of PE with FGR.

Exposure to omentum adipose tissue conditioned medium from obese pregnant women promotes myometrial artery dysfunction

Underlying mechanisms of poor pregnancy outcome in obese (OB) mothers (body mass index [BMI] ≥ 30 kg/m2) are unknown. Our studies demonstrate that OB pregnant women have altered myometrial artery (MA) function related to the thromboxane and nitric oxide pathways. In obesity, increased central fat mass is associated with an altered endocrine milieu. We tested the hypothesis that in OB pregnant women the omentum, a central fat store, releases factors that promote dysfunction in normal MAs.

Pomegranate Juice Supplementation Alters Utero-Placental Vascular Function and Fetal Growth in the eNOS−/− Mouse Model of Fetal Growth Restriction

The eNOS−/− mouse provides a well-characterized model of fetal growth restriction (FGR) with altered uterine and umbilical artery function and reduced utero- and feto-placental blood flow. Pomegranate juice (PJ), which is rich in antioxidants and bioactive polyphenols, has been posited as a beneficial dietary supplement to promote cardiovascular health. We hypothesized that maternal supplementation with PJ will improve uterine and umbilical artery function and thereby enhance fetal growth in the eNOS−/− mouse model of FGR. Wild type (WT, C57Bl/6J) and eNOS−/− mice were supplemented from E12.5-18.5 with either PJ in their drinking water or water alone. At E18.5 uterine (UtA) and umbilical (UmbA) arteries were isolated for study of vascular function, fetuses and placentas were weighed and fetal biometric measurements taken. PJ supplementation significantly increased UtA basal tone (both genotypes) and enhanced phenylephrine-induced contraction in eNOS−/− but not WT mice. Conversely PJ significantly reduced UtA relaxation in response to both acetylcholine (Ach) and sodium nitroprusside (SNP), endothelium dependent and independent vasodilators respectively from WT but not eNOS−/− mice. UmbA sensitivity to U46619-mediated contraction was increased by PJ supplementation in WT mice; PJ enhanced contraction and relaxation of UmbA to Ach and SNP respectively in both genotypes. Contrary to our hypothesis, the changes in artery function induced by PJ were not associated with an increase in fetal weight. However, PJ supplementation reduced litter size and fetal abdominal and head circumference in both genotypes. Collectively the data do not support maternal PJ supplementation as a safe or effective treatment for FGR.