Mark S. Longtine

Vitamin D effects on pregnancy and the placenta.

Vitamin D is a pleiotropic secosteroid hormone important for health and disease prevention. The actions of vitamin D are mediated by the vitamin D receptor that binds the active form of vitamin D [1,25(OH)(2)D] to induce both transcriptional and non-genomic responses. Vitamin D has well known classical functions in calcium uptake and bone metabolism, but more recent work highlights the importance of the nonclassical actions of vitamin D in a variety of cell types. These actions include modulation of the innate and adaptive immune systems and regulation of cell proliferation. Adequate vitamin D intake is essential for maternal and fetal health during pregnancy, and epidemiological data indicate that many pregnant women have sub-optimal vitamin D levels. Notably, vitamin D deficiency correlates with preeclampsia, gestational diabetes mellitus, and bacterial vaginosis, and an increased risk for C-section delivery. Recent work emphasizes the importance of nonclassical roles of vitamin D in pregnancy and the placenta. The placenta produces and responds to vitamin D where vitamin D functions as a modulator of implantation, cytokine production and the immune response to infection. We describe vitamin D metabolism and the cellular responses to vitamin D, and then summarize the role of vitamin D in placental trophoblast, pregnancy and the fetus.

Placental dysfunction and fetal programming: the importance of placental size, shape, histopathology, and molecular composition.

Normal function of the placenta is pivotal for optimal fetal growth and development. Fetal programming commonly is associated with placental dysfunction that predisposes to obstetric complications and suboptimal fetal outcomes. We consider several clinical phenotypes for placental dysfunction that likely predispose to fetal programming. Some of these reflect abnormal development of the chorioallantoic placenta in size, shape, or histopathology. Others result when exogenous stressors in the maternal environment combine with maladaptation of the placental response to yield small placentas with limited reserve, as typical of early-onset intrauterine growth restriction and preeclampsia. Still others reflect epigenetic changes, including altered expression of imprinted genes, altered enzymatic activity, or altered efficiencies in nutrient transport. Although the human placenta is a transient organ that persists only 9 months, the effects of this organ on the offspring remain for a lifetime.

Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts

The human placenta is key to pregnancy outcome, and the elevated oxidative stress present in many complicated pregnancies contributes to placental dysfunction and suboptimal pregnancy outcomes. We tested the hypothesis that pomegranate juice, which is rich in polyphenolic antioxidants, limits placental trophoblast injury in vivo and in vitro. Pregnant women with singleton pregnancies were randomized at 35∼38 wk gestation to 8 oz/day of pomegranate juice or apple juice (placebo) until the time of delivery. Placental tissues from 12 patients (4 in the pomegranate group and 8 in the control group) were collected for analysis of oxidative stress. The preliminary in vivo results were extended to oxidative stress and cell death assays in vitro. Placental explants and cultured primary human trophoblasts were exposed to pomegranate juice or glucose (control) under defined oxygen tensions and chemical stimuli. We found decreased oxidative stress in term human placentas from women who labored after prenatal ingestion of pomegranate juice compared with apple juice as control. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in term villous explants and primary trophoblast cultures exposed to hypoxia, the hypoxia mimetic cobalt chloride, and the kinase inhibitor staurosporine. Punicalagin, but not ellagic acid, both prominent polyphenols in pomegranate juice, reduced oxidative stress and stimulus-induced apoptosis in cultured syncytiotrophoblasts. We conclude that pomegranate juice reduces placental oxidative stress in vivo and in vitro while limiting stimulus-induced death of human trophoblasts in culture. The polyphenol punicalagin mimics this protective effect. We speculate that antenatal intake of pomegranate may limit placental injury and thereby may confer protection to the exposed fetus.

Caspase-mediated apoptosis of trophoblasts in term human placental villi is restricted to cytotrophoblasts and absent from the multinucleated syncytiotrophoblast

Human placental villi are surfaced by a multinucleated and terminally differentiated epithelium, the syncytiotrophoblast, with a subjacent layer of mononucleated cytotrophoblasts that can divide and fuse to replenish the syncytiotrophoblast. The objectives of this study were i) to develop an approach to definitively identify and distinguish cytotrophoblasts from the syncytiotrophoblast, ii) to unambiguously determine the relative susceptibility of villous cytotrophoblasts and syncytiotrophoblast to constitutive and stress-induced apoptosis mediated by caspases, and iii) to understand the progression of apoptosis in villous trophoblasts. Confocal microscopy with co-staining for E-cadherin and DNA allowed us to clearly distinguish the syncytiotrophoblast from cytotrophoblasts and identified that many cytotrophoblasts are deeply interdigitated into the syncytiotrophoblast. Staining for specific markers of caspase-mediated apoptosis indicate that apoptosis occurs readily in cytotrophoblasts but is remarkably inhibited in the syncytiotrophoblast. To determine if an apoptotic cell or cell fragment was from a cytotrophoblast or syncytiotrophoblast, we found co-staining with E-cadherin along with a marker for apoptosis was essential: in the absence of E-cadherin staining, apoptotic cytotrophoblasts would easily be mistaken as representing localized regions of apoptosis in the syncytiotrophoblast. Regions with perivillous fibrin-containing fibrinoid contain the remnants of trophoblast apoptosis, and we propose this apoptosis occurs only after physical isolation of a region of the syncytium from the main body of the syncytium. We propose models for the progression of apoptosis in villous cytotrophoblasts and for why caspase-mediated apoptosis does not occur within the syncytium of placental villi.

Hypoxia downregulates p53 but induces apoptosis and enhances expression of BAD in cultures of human syncytiotrophoblasts

Hypoxia is commonly assigned a role in the placental dysfunction characteristic of preeclampsia and intrauterine growth restriction. We previously showed that hypoxia upregulates p53 and enhances apoptosis in primary cultures of human cytotrophoblasts. Here we tested the hypothesis that hypoxia also induces apoptosis in syncytiotrophoblasts by upregulation of p53. Primary cultures of human cytotrophoblasts that had differentiated into syncytiotrophoblasts by 52 h were exposed for ≤24 h to 20% or <1% oxygen in the presence or absence of staurosporine or the p53 modulators nutlin-3, pifithrin-α, and pifithrin-μ. Proteins were detected by Western blot analysis or immunofluorescence. Compared with 20% oxygen, exposure of syncytiotrophoblasts to <1% oxygen upregulated hypoxia-inducible factor (HIF)-1α and rapidly downregulated p53. Activity of p53 in hypoxic syncytiotrophoblasts was reduced by the higher expression of the negative p53 regulator MDMX and by the reduction of phosphorylation of p53 at Ser(392), which reduces p53 activity. Conversely, staurosporine, a kinase inhibitor, and nutlin-3, a drug that enhances p53 expression, both raised p53 levels and increased the rate of apoptosis in syncytiotrophoblasts compared with vehicle controls. Immunofluorescence staining showed p53 immunolocalized to both cytoplasm and nuclei of nutlin-3-exposed syncytiotrophoblasts. The hypoxia-induced apoptosis in syncytiotrophoblasts correlated with enhanced expression of the proapoptotic BAD and a reduced level of antiapoptotic BAD phosphorylated on Ser(112). We surmise that cell death induced by extreme hypoxia in syncytiotrophoblasts follows a non-p53-dependent pathway, unlike that of a nonhypoxic stimulus and unlike hypoxic cytotrophoblasts. We speculate that downregulation of p53 activity in response to hypoxia reduces or eliminates the apoptosis transduced by the p53 pathway in syncytiotrophoblasts, thereby limiting cell death and maintaining the integrity of this critical villous component.

The Human Placental Sexome Differs between Trophoblast Epithelium and Villous Vessel Endothelium

Molecular mechanisms underlying sexual dimorphism in mammals, fetal sex influences on intrauterine development, and the sex-biased susceptibility for selected diseases in adulthood are novel areas of current research. As importantly, two decades of multifaceted research has established that susceptibility to many adult disorders originates in utero, commonly secondary to the effects of placental dysfunction. We hypothesized that fetal sex influences gene expression and produces functional differences in human placentas. We thus extended previous studies on sexual dimorphism in mammals, which used RNA isolated from whole tissues, to investigate the effects of sex on four cell-phenotypes within a single key tissue, human placental villi. The cells studied included cytotrophoblasts, syncytiotrophoblast, arterial and venous endothelial cells. The cells were isolated from placentas of male or female fetuses and subjected to microarray analysis. We found that fetal sex differentially affected gene expression in a cell-phenotype dependent manner among all four cell-phenotypes. The markedly enriched pathways in males were identified to be signaling pathways for graft-versus-host disease as well as the immune and inflammatory systems that parallel the reported poorer outcome of male fetuses. Our study is the first to compare global gene expression by microarray analysis in purified, characterized, somatic cells from a single human tissue, i.e. placental villi. Importantly, our findings demonstrate that there are cell-phenotype specific, and tissue-specific, sex-biased responses in the human placenta, suggesting fetal sex should be considered as an independent variable in gene expression analysis of human placental villi.

Punicalagin, a polyphenol in pomegranate juice, downregulates p53 and attenuates hypoxia-induced apoptosis in cultured human placental syncytiotrophoblasts

Oxidative stress is associated with placental dysfunction and suboptimal pregnancy outcomes. Therapeutic interventions to limit placental injury from oxidative stress are lacking. Punicalagin is an ellagitannin and a potent antioxidant in pomegranate juice. We showed that both pomegranate juice and punicalagin decrease oxidative stress and apoptosis in cultured syncytiotrophoblasts. p53 is involved in the oxidative stress-induced apoptosis in trophoblasts. We now test the hypothesis that punicalagin limits trophoblast injury in vitro by regulating the levels of p53. We examined the expression of p53, mouse double minute 2 homolog, p21, hypoxia-inducible factor (HIF) α, and selected members of the B cell lymphoma 2 (BCL2) family of proteins in cultured syncytiotrophoblasts exposed to ≤1% oxygen in the absence or presence of punicalagin. We found that punicalagin attenuated hypoxia-induced apoptosis in syncytiotrophoblasts, as quantified by levels of cleaved poly-ADP ribose polymerase. This protective effect was in part mediated by reduced p53 activity shown by decreased expression of p21, lower HIF1α expression, and limited activity of caspases 9 and 3. There was no change in expression of proteins in the BCL2 family, which are also important in apoptosis. The data support a role for downregulation of p53 in the protection of human trophoblasts by punicalagin.

Live-cell imaging shows apoptosis initiates locally and propagates as a wave throughout syncytiotrophoblasts in primary cultures of human placental villous trophoblasts

Human placental villi are surfaced by the syncytiotrophoblast, a multinucleated, epithelial-cell layer that functions in maternal-fetal exchange. Mononucleated cytotrophoblasts are subjacent to the syncytiotrophoblast. Using confocal fluorescence microscopy of third-trimester villi, we previously found that cytotrophoblasts are often interdigitated into the syncytiotrophoblast, that cytotrophoblasts undergo caspase-mediated apoptosis, and that apoptosis is much lower, and perhaps completely inhibited, in intact syncytiotrophoblast lacking fibrin-type fibrinoid. Previous analysis of primary cultures of human trophoblasts also indicated lower levels of apoptosis in syncytiotrophoblast compared to cytotrophoblasts. Here, using confocal microscopy we find that cultured cytotrophoblasts and syncytiotrophoblasts display complex structural relationships, as in vivo, and that apoptosis of a cytotrophoblast or syncytiotrophoblast does not induce apoptosis of adjacent trophoblasts. Using live-cell imaging of mitochondrial depolarization and nuclear condensation in cultured syncytiotrophoblasts, we show apoptosis initiates in a localized region and propagates radially at ∼5 μm/min with no loss of velocity until the entire syncytium has undergone apoptosis. The rate of propagation is similar in cases of spontaneous apoptosis and in apoptosis that occurs in the presence of cobalt chloride or rotenone, two inducers of apoptosis. We suggest that inhibition of syncytiotrophoblast apoptosis in vivo is important to prevent widespread syncytiotrophoblast death, which would result in placental dysfunction and contribute to poor pregnancy outcomes.

Pericellular oxygen concentration of cultured primary human trophoblasts.

INTRODUCTION Oxygen is pivotal in placental development and function. In vitro culture of human trophoblasts provides a useful model to study this phenomenon, but a hotly debated issue is whether or not the oxygen tension of the culture conditions mimics in vivo conditions. We tested the hypothesis that ambient oxygen tensions in culture reflect the pericellular oxygen levels. METHODS We used a microelectrode oxygen sensor to measure the concentration of dissolved oxygen in the culture medium equilibrated with 21%, 8% or <0.5% oxygen. RESULTS The concentration of oxygen in medium without cells resembled that in the ambient atmosphere. The oxygen concentration present in medium bathing trophoblasts was remarkably dependent on the depth within the medium where sampling occurred, and the oxygen concentration within the overlying atmosphere was not reflected in medium immediately adjacent to the cells. Indeed, the pericellular oxygen concentration was in a range that most would consider severe hypoxia, at ≤0.6% oxygen or about 4.6 mm Hg, when the overlying atmosphere was 21% oxygen. CONCLUSIONS We conclude that culture conditions of 21% oxygen are unable to replicate the pO(2) of 40-60 mm Hg commonly attributed to the maternal blood in the intervillous space in the second and third trimesters of pregnancy. We further surmise that oxygen atmospheres in culture conditions between 0.5% and 21% provide different oxygen fluxes in the immediate pericellular environment yet can still yield insights into the responses of human trophoblast to different oxygen conditions.

N-myc downstream-regulated gene 1 (NDRG1) mediates pomegranate juice protection from apoptosis in hypoxic BeWo cells but not in primary human trophoblasts

INTRODUCTION N-Myc downstream-regulated gene 1 (NDRG1) expression is increased in placentas of human pregnancies with intrauterine growth restriction and in hypoxic cultured primary trophoblasts. We previously showed that elevated NDRG1 decreases trophoblast apoptosis induced by hypoxia. Separately, we found that pomegranate juice (PJ) decreases cell death induced by hypoxia in trophoblasts. Here, we test the hypothesis that PJ protects trophoblasts from hypoxia-induced apoptosis by modulating NDRG1 expression. METHODS Quantitative rtPCR was used to investigate the effects of PJ treatment on mRNA levels of 22 candidate genes involved in apoptosis, oxidative stress, and differentiation in trophoblasts. Western blotting and immunofluorescence were used to analyze NDRG1 protein levels. siRNA-mediated NDRG1 knockdown was used to investigate the role of NDRG1 in response to PJ in hypoxic BeWo choriocarcinoma cells and hypoxic cultured primary human trophoblasts. RESULTS The mRNA levels of eight genes were altered, with NDRG1 showing the largest response to PJ and thus, we pursued the role of NDRG1 here. PJ significantly increased NDRG1 protein expression in primary trophoblasts and in BeWo cells. Knockdown of NDRG1 in hypoxic BeWo cells in the presence of PJ yielded increased apoptosis. In contrast, knockdown of NDRG1 in hypoxic primary trophoblasts in the presence of PJ did not increase apoptosis. DISCUSSION We conclude that the PJ-mediated decrease in cell death in hypoxia is partially mediated by NDRG1 in BeWo cells but not in primary trophoblasts. The disparate effects of NDRG1 between BeWo cells and primary trophoblasts indicate caution is required when extrapolating from results obtained with cell lines to primary trophoblasts.