Lewis Renshall

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.

Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction.

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-&agr;–induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

Crossing mice deficient in eNOS with placental-specific Igf2 knockout mice: A new model of fetal growth restriction

We tested the hypothesis that crossing two mouse models of fetal growth restriction (FGR) of differing phenotype would induce more severe FGR than either model alone. Female endothelial nitric oxide synthase knockout mice (eNOS−/−) were mated with placental-specific Igf2 knockout males (P0). Resultant fetuses were no more growth restricted than those with P0 deletion alone. However, P0 deletion attenuated the reduced placental system A amino acid transporter activity previously observed in eNOS−/− mice. Manipulating maternal and fetal genotypes provides a means to compare maternal and fetal regulation of fetal growth.

In vitro assessment of mouse fetal abdominal aortic vascular function

Fetal growth restriction (FGR) affects 3–8% of human pregnancies. Mouse models have provided important etiological data on FGR; they permit the assessment of treatment strategies on the physiological function of both mother and her developing offspring. Our study aimed to 1) develop a method to assess vascular function in fetal mice and 2) as a proof of principle ascertain whether a high dose of sildenafil citrate (SC; Viagra) administered to the pregnant dam affected fetal vascular reactivity. We developed a wire myography methodology for evaluation of fetal vascular function in vitro using the placenta-specific insulin-like growth factor II (Igf2) knockout mouse (P0; a model of FGR). Vascular function was determined in abdominal aortas isolated from P0 and wild-type (WT) fetuses at embryonic day (E) 18.5 of gestation. A subset of dams received SC 0.8 mg/ml via drinking water from E12.5; data were compared with water-only controls. Using wire myography, we found that fetal aortic rings exhibited significant agonist-induced contraction, and endothelium-dependent and endothelium-independent relaxation. Sex-specific alterations in reactivity were noted in both strains. Maternal treatment with SC significantly attenuated endothelium-dependent and endothelium-independent relaxation of fetal aortic rings. Mouse fetal abdominal aortas reproducibly respond to vasoactive agents. Study of these vessels in mouse genetic models of pregnancy complications may 1) help to delineate early signs of abnormal vascular reactivity and 2) inform whether treatments given to the mother during pregnancy may impact upon fetal vascular function.

Adaptations in Maternofetal Calcium Transport in Relation to Placental Size and Fetal Sex in Mice

Appropriate placental transport of calcium is essential for normal fetal skeletal mineralization. In fetal growth restriction (FGR), the failure of a fetus to achieve its growth potential, a number of placental nutrient transport systems show reduced activity but, in the case of calcium, placental transport is increased. In a genetic mouse model of FGR this increase, or adaptation, maintains appropriate fetal calcium content, relative to the size of the fetus, despite a small, dysfunctional placenta. It is unknown whether such an adaptation is also apparent in small, but normally functioning placentas. We tested the hypothesis that calcium transfer would be up-regulated in the lightest vs. heaviest placentas in the same C57Bl/6J wild-type (WT) mouse litter. Since lightest placentas are often from females, we also assessed whether fetal sex influenced placental calcium transfer. Placentas and fetuses were collected at embryonic day (E)16.5 and 18.5; the lightest and heaviest placentas, and female and male fetuses, were identified. Unidirectional maternofetal calcium clearance (CaKmf) was assessed following 45Ca administration to the dam and subsequent radiolabel counts within the fetuses. Placental expression of calcium pathway components was measured by Western blot. Data (median) are lightest placenta expressed as percentage of the heaviest within a litter and analyzed by Wilcoxon signed-rank test. In WT mice having normally grown fetuses, CaKmf, per gram placenta near term, in the lightest placentas was increased (126%; P < 0.05) in association with reduced fetal calcium accretion earlier in gestation (92%; P < 0.05), that was subsequently normalized near term. Increased placental expression of calbindin-D9K, an important calcium binding protein, was observed in the lightest placentas near term (122%; P < 0.01). There was no difference in fetal calcium accretion between male and female littermates but a trend toward higher CaKmf in females (P = 0.055). These data suggest a small, normal placenta adapts calcium transfer according to its size, as previously demonstrated in a mouse model of FGR. Fetal sex had limited influence on this adaptive increase. These adaptations are potentially driven by fetal nutrient demand, as evidenced by the normalization of fetal calcium content. Understanding the regulatory mechanisms involved may provide novel avenues for treating placental dysfunction.

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.

Melatonin Increases Fetal Weight in Wild-Type Mice but Not in Mouse Models of Fetal Growth Restriction

Fetal growth restriction (FGR) presents with an increased risk of stillbirth and childhood and adulthood morbidity. Melatonin, a neurohormone and antioxidant, has been suggested as having therapeutic benefit in FGR. We tested the hypothesis that melatonin would increase fetal growth in two mouse models of FGR which together represent a spectrum of the placental phenotypes in this complication: namely the endothelial nitric oxide synthase knockout mouse (eNOS-/-) which presents with abnormal uteroplacental blood flow, and the placental specific Igf2 knockout mouse (P0+/-) which demonstrates aberrant placental morphology akin to human FGR. Melatonin (5 μg/ml) was administered via drinking water from embryonic day (E)12.5 in C57Bl/6J wild-type (WT), eNOS-/-, and P0+/- mice. Melatonin supplementation significantly increased fetal weight in WT, but not eNOS-/- or P0+/- mice at E18.5. Melatonin did, however, significantly increase abdominal circumference in P0+/- mice. Melatonin had no effect on placental weight in any group. Uterine arteries from eNOS-/- mice demonstrated aberrant function compared with WT but melatonin treatment did not affect uterine artery vascular reactivity in either of these genotypes. Umbilical arteries from melatonin treated P0+/- mice demonstrated increased relaxation in response to the nitric oxide donor SNP compared with control. The increased fetal weight in WT mice and abdominal circumference in P0+/-, together with the lack of any effect in eNOS-/-, suggest that the presence of eNOS is required for the growth promoting effects of melatonin. This study supports further work on the possibility of melatonin as a treatment for FGR.