Estrogen stimulates dimethylarginine dimethylaminohydrolase activity and the metabolism of asymmetric dimethylarginine.

Background— Experimental evidence suggests that estrogens stimulate the production of nitric oxide (NO) by vascular endothelial cells. This effect has been attributed to increased expression and enzymatic activity of both the constitutive and inducible isoforms of NO synthase. In this study, we have investigated whether estrogens regulate the metabolism or release of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase. Methods and Results— The concentration of ADMA in the plasma of 15 postmenopausal women was 0.722±0.04 μmol/L (mean±SEM). Two weeks after subcutaneous implantation with estradiol, there was an increase in plasma estradiol concentration from 0.693±0.075 to 0.81±87 nmol/L, which was accompanied by a significant fall in plasma ADMA concentration to 0.588±0.03 μmol/L (P=0.006). Human and murine endothelial cell lines previously cultured in estrogen-free medium and then exposed to 17β-estradiol showed a dose-dependent decrease in the release of ADMA. This reached statistical significance at 10−14 mol/L 17β-estradiol and was accompanied by a corresponding increase in the activity of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catalyzes the metabolism of ADMA. Conclusions— We have demonstrated that estrogens can alter the catabolism and release of ADMA in vitro and reduce the circulating concentration in vivo. We therefore propose that increased DDAH activity and the subsequent fall in ADMA could contribute to the positive effect of estrogen on NO synthesis

Strategies for investigating the maternal-fetal interface in the first trimester of pregnancy: What can we learn about pathology?

The pathologies of the pregnancy complications pre-eclampsia (PE) and fetal growth restriction (FGR) are established in the first trimester of human pregnancy. In a normal pregnancy, decidual spiral arteries are transformed into wide diameter, non-vasoactive vessels capable of meeting the increased demands of the developing fetus for nutrients and oxygen. Disruption of this transformation is associated with PE and FGR. Very little is known of how these first trimester changes are regulated normally and even less is known about how they are compromised in complicated pregnancies. Interactions between maternal and placental cells are essential for pregnancy to progress and this review will summarise the challenges in investigating this area. We will discuss how first trimester studies of pregnancies with an increased risk of developing PE/FGR have started to provide valuable information about pregnancy at this most dynamic and crucial time. We will discuss where there is scope to progress these studies further by refining the ability to identify compromised pregnancies at an early stage, by integrating information from many cell types from the same pregnancy, and by improving our methods for modelling the maternal-fetal interface in vitro

Fetal-derived trophoblast use the apoptotic cytokine tumor necrosis factor-alpha-related apoptosis-inducing ligand to induce smooth muscle cell death.

Remodeling of the uterine spiral arteries during pregnancy transforms them from high to low resistance vessels that lack vasoconstrictive properties. This process is essential to meet the demand for increased blood flow imposed by the growing fetus. Loss of endothelial and smooth muscle cells (SMC) is evident in remodeled arteries but the mechanisms underlying this transformation remain unknown. This study investigated the hypothesis that fetal trophoblast invading from the placenta instigate remodeling by triggering cell death in vascular SMC. Specifically, a role for trophoblast-derived death inducing cytokine tumor necrosis factor-α–related apoptosis-inducing ligand (TRAIL) was investigated. Expression of the activating TRAIL receptors R1 and R2 was detected by flow cytometry on human aortic SMC and by immunohistochemistry on spiral artery SMC. Recombinant human TRAIL induced human aortic SMC apoptosis, which was inhibited by antibodies against TRAIL-R1 or -R2. Perfusion of denuded spiral artery segments with recombinant human TRAIL also induced SMC apoptosis. Trophoblasts isolated from first trimester placenta expressed membrane-associated TRAIL and induced apoptosis of human aortic SMC; apoptosis was significantly inhibited by a recombinant human TRAIL-R1:Fc construct. Trophoblast within the first trimester placental bed also expressed TRAIL. These data show that: 1) TRAIL causes SMC death; 2) trophoblast produce the apoptotic cytokine TRAIL; and 3) trophoblast induce SMC apoptosis via a TRAIL-dependent mechanism. We conclude that TRAIL produced by trophoblast causes apoptosis of SMC and thus may contribute to SMC loss during spiral artery remodeling in pregnancy

Uterine spiral artery remodeling involves endothelial apoptosis induced by extravillous trophoblasts through Fas/FasL interactions.

Objective— Invasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved. Methods and Results— Spiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model. Conclusions— Extravillous trophoblast induction of endothelial apoptosis is a possible mechanism by which the endothelium is removed, and vascular remodeling may occur in uterine spiral arteries. Fas/FasL interactions have an important role in trophoblast-induced endothelial apoptosis

Impaired decidual natural killer cell regulation of vascular remodelling in early human pregnancies with high uterine artery resistance

During human pregnancy, natural killer (NK) cells accumulate in the maternal decidua, but their specific roles remain to be determined. Decidual NK (dNK) cells are present during trophoblast invasion and uterine spiral artery remodelling. These events are crucial for successful placentation and the provision of an adequate blood supply to the developing fetus. Remodelling of spiral arteries is impaired in the dangerous pregnancy complication pre-eclampsia. We studied dNK cells isolated from pregnancies at 9-14 weeks' gestation, screened by uterine artery Doppler ultrasound to determine resistance indices which relate to the extent of spiral artery remodelling. dNK cells were able to promote the invasive behaviour of fetal trophoblast cells, partly through HGF. Cells isolated from pregnancies with higher resistance indices were less able to do this and secreted fewer pro-invasive factors. dNK cells from pregnancies with normal resistance indices could induce apoptotic changes in vascular smooth muscle and endothelial cells in vitro, events of importance in vessel remodelling, partly through Fas signalling. dNK cells isolated from high resistance index pregnancies failed to induce vascular apoptosis and secreted fewer pro-apoptotic factors. We have modelled the cellular interactions at the maternal-fetal interface and provide the first demonstration of a functional role for dNK cells in influencing vascular cells. A potential mechanism contributing to impaired vessel remodelling in pregnancies with a higher uterine artery resistance is presented. These findings may be informative in determining the cellular interactions contributing to the pathology of pregnancy disorders where remodelling is impaired, such as pre-eclampsia. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Decidual cell regulation of trophoblast is altered in pregnancies at risk of pre-eclampsia

Successful implantation and placentation are dependent on the interaction between decidual stromal cells (DSC) and extravillous trophoblast (EVT) cells. The extent of trophoblast invasion relies on communication between the placenta and maternal decidua. The cyclical process of decidualisation induces a transformation of endometrial fibroblasts to secretory DSC; these secreted products have many functions including the control of trophoblast invasion. Inadequate trophoblast invasion and remodelling of the uterine vessels (the spiral arteries) are associated with pregnancy disorders such as pre-eclampsia. Uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure of remodelling. DSC were isolated from pregnancies with normal (normal RI) or impaired (high RI) spiral artery remodelling. Following isolation, DSC were re-decidualised using cAMP and MPA and secretion of the decidualisation markers IGFBP-1 and prolactin assessed. We examined the impact of DSC secreted factors on trophoblast cell function, using the EVT cell line SGHPL-4. We demonstrated that DSC exposed to decidual factors were able to re-decidualise in vitro and that the chemoattraction of trophoblasts by DSC is impaired in pregnancies with high RI. This study provides new insights into the role that DSC play in regulating EVT functions during the first trimester of pregnancy. This is the first study to demonstrate that DSC from pregnancies with impaired vascular remodelling in the first trimester secrete factors that inhibit the directional movement of trophoblast cells. This finding may be important in understanding aberrant trophoblast invasion in pregnancies where vascular remodelling is impaired

Trophoblast-induced changes in C-x-C motif chemokine 10 expression contribute to vascular smooth muscle cell dedifferentiation during spiral artery remodeling.

Objective- During pregnancy, fetal trophoblast disrupt endothelial cell and vascular smooth muscle cell (VSMC) interactions in spiral arteries of the maternal decidua to enable increased nutritional and oxygen delivery to the fetus. Little is known regarding this transformation because of difficulties of studying human pregnancy in vivo. This study investigated how trophoblast-secreted factors affect the interactions of vascular cells and the differentiation status of VSMC during spiral arteries remodeling using 3-dimensional vascular spheroid coculture. Methods and Results— Endothelial cell and VSMC were cocultured in hanging droplets to form spheroids representing an inverted vessel lumen. Control or conditioned media from an extravillous trophoblast (EVT) cell line was incubated with vascular spheroids for 24 hours. Spheroid RNA was then analyzed by Illumina Sentrix BeadChip array. Spheroids incubated with EVT conditioned medium showed significant up/downregulation of 101 genes (>1.5-fold; P<0.05), including an upregulation of C-X-C motif chemokine 10 (IP-10). C-X-C motif chemokine 10 expression was confirmed by qualitative real-time PCR and Western blot analysis of spheroids, and immunohistochemistry of first trimester decidua and ex vivo dissected nonplacental bed spiral arteries. EVT conditioned medium reduced VSMC expression of differentiation markers, and both EVT conditioned medium and C-X-C motif chemokine 10 increased motility of VSMC indicating dedifferentiation of VSMC. Conclusion— EVT-induced C-X-C motif chemokine 10 expression may contribute to spiral arteries remodeling during pregnancy by altering the motility and differentiation status of the VSMC in the vessel

Inhibition of Trophoblast-Induced Spiral Artery Remodeling Reduces Placental Perfusion in Rat Pregnancy.

Rats harboring the human angiotensinogen and human renin genes develop preeclamptic features in pregnancy. The preeclamptic rats exhibit a deeper trophoblast invasion associated with a reduced resistance index by uterine Doppler. Doxycycline inhibits matrix metalloproteinase activity. We tested the hypothesis that matrix metalloproteinase inhibition reduces trophoblast invasion with subsequent changes in placental perfusion. Preeclamptic and pregnant control Sprague-Dawley rats were treated with doxycycline (30 mg/kg of body weight orally) from gestational day 12 until day 18. Placental perfusion was assessed using a micromarker contrast agent. The animals were euthanized on day 18 of pregnancy; biometric data were acquired, and trophoblast invasion was analyzed. Doxycycline resulted in intrauterine growth retardation and lighter placentas in both groups. Maternal body weight was not affected. As shown earlier, preeclamptic rats exhibited a deeper endovascular trophoblast invasion. However, doxycycline treatment reduced trophoblast invasion in the preeclamptic rats. The physiological spiral artery remodeling, as assessed by the deposition of fibrinoid and α-actin in the spiral artery contour, was significantly reduced by doxycycline. The vascularity index, as assessed by perfusion measurement of the placenta, was reduced after doxycycline treatment in preeclamptic rats. Thus, matrix metalloproteinase inhibition with doxycycline leads to reduced trophoblast invasion and associated reduced placental perfusion. These studies are the first to show that reducing trophoblast-induced vascular remodeling decreases subsequent placental perfusion. Our model allows the study of dysregulated trophoblast invasion and vascular remodeling in vivo to gain important insights into preeclampsia-related mechanisms

Effects of Circulating and Local Uteroplacental Angiotensin II in Rat Pregnancy.

The renin-angiotensin (Ang) system is important during placental development. Dysregulation of the renin-Ang system is important in preeclampsia (PE). Female rats transgenic for the human angiotensinogen gene crossed with males transgenic for the human renin gene develop the PE syndrome, whereas those of the opposite cross do not. We used this model to study the role of Ang II in trophoblast invasion, which is shallow in human PE but deeper in this model. We investigated the following groups: PE rats, opposite-cross rats, Ang II–infused rats (1000 ng/kg per day), and control rats. Ang II infusion increased only circulating Ang II levels (267.82 pg/mL), opposite cross influenced only uteroplacental Ang II (13.52 fmol/mg of protein), and PE increased both circulating (251.09 pg/mL) and uteroplacental (19.24 fmol/mg of protein) Ang II. Blood pressure and albuminuria occurred in the models with high circulating Ang II but not in the other models. Trophoblast invasion increased in PE and opposite-cross rats but not in Ang II–infused rats. Correspondingly, uterine artery resistance index increased in Ang II–infused rats but decreased in PE rats. We then studied human trophoblasts and villous explants from first-trimester pregnancies with time-lapse microscopy. Local Ang II dose-dependently increased migration by 75%, invasion by 58%, and motility by 282%. The data suggest that local tissue Ang II stimulates trophoblast invasion in vivo in the rat and in vitro in human cells, a hitherto fore unrecognized function. Conceivably, upregulation of tissue Ang II in the maternal part of the placenta represents an important growth factor for trophoblast invasion and migration