Placental Tissue Explants Research Articles

S17-01: Human placental villous tissue explants in toxicology

S17-01: Human placental villous tissue explants in toxicology

Nitric Oxide Affects Heme Oxygenase-1, Hepcidin, and Transferrin Receptor Expression in the Placenta.

Nitric oxide (NO) is a gasotransmitter that avidly binds both free and heme-bound iron, forming relatively stable iron nitrosyl compounds (FeNOs). We have previously demonstrated that FeNOs are present in the human placenta and are elevated in preeclampsia and intrauterine growth restriction. The ability of NO to sequester iron raises the possibility of the NO-mediated disruption of iron homeostasis in the placenta. In this work, we tested whether exposure of placental syncytiotrophoblasts or villous tissue explants to sub-cytotoxic concentrations of NO would elicit the formation of FeNOs. Furthermore, we measured changes in the mRNA and protein expression levels of key iron regulatory genes in response to NO exposure. Ozone-based chemiluminescence was used to measure concentrations of NO and its metabolites. Our results showed a significant increase in FeNO levels in placental cells and explants treated with NO (p < 0.0001). The mRNA and protein levels of HO-1 were significantly increased in both cultured syncytiotrophoblasts and villous tissue explants (p < 0.01), and the mRNA levels of hepcidin and transferrin receptor were significantly increased in culture syncytiotrophoblasts and villous tissue explants, respectively, (p < 0.01), while no changes were seen in the expression levels of divalent metal transporter-1 or ferroportin. These results suggest a potential role for NO in iron homeostasis in the human placenta and could be relevant for disorders of pregnancy such as fetal growth restriction and preeclampsia.

Toxicity of anticancer drugs in human placental tissue explants and trophoblast cell lines

The application of anticancer drugs during pregnancy is associated with placenta-related adverse pregnancy outcomes. Therefore, it is important to study placental toxicity of anticancer drugs. The aim of this study was to compare effects on viability and steroidogenesis in placental tissue explants and trophoblast cell lines. Third trimester placental tissue explants were exposed for 72 h (culture day 4–7) to a concentration range of doxorubicin, paclitaxel, cisplatin, carboplatin, crizotinib, gefitinib, imatinib, or sunitinib. JEG-3, undifferentiated BeWo, and syncytialised BeWo cells were exposed for 48 h to the same drugs and concentrations. After exposure, tissue and cell viability were assessed and progesterone and estrone levels were quantified in culture medium. Apart from paclitaxel, all compounds affected both cell and tissue viability at clinically relevant concentrations. Paclitaxel affected explant viability moderately, while it reduced cell viability by 50% or more in all cell lines, at 3–10 nM. Doxorubicin (1 µM) reduced viability in explants to 83 ± 7% of control values, whereas it fully inhibited viability in all cell types. Interference with steroid release in explants was difficult to study due to large variability in measurements, but syncytialised BeWo cells proved suitable for this purpose. We found that 1 µM sunitinib reduced progesterone release to 76 ± 6% of control values, without affecting cell viability. While we observed differences between the models for paclitaxel and doxorubicin, most anticancer drugs affected viability significantly in both placental explants and trophoblast cell lines. Taken together, the placenta should be recognized as a potential target organ for toxicity of anticancer drugs.

Efficient enhancement of capsaicinoids biosynthesis in cell suspension cultures of Capsicum chinense Jacq. cv. ‘Umorok’ by elicitors and differential gene expression analysis of elicited cultures

Capsaicinoids are responsible for pungency in chillies and have multiple health benefits and industrial applications. In vitro cell cultures of Capsicum with high capsaicinoids content may have the potential to serve as an alternative source for production of capsaicinoids. The present study investigated the effects of various elicitors, precursors and intermediates on the production of capsaicinoids in cell suspension cultures established from callus derived from the placental tissue explants of the highly pungent chilli cultivar, Capsicum chinense Jacq. cv. ‘Umorok’. The study also, for the first time, reports the effect of elicitors on expression patterns of capsaicinoids biosynthesis genes in cell suspension cultures of C. chinense by quantitative real-time PCR. The maximum capsaicinoids content was obtained in cells elicitated with chitosan with average capsaicin and dihydrocapsaicin contents of 2.87 mg g−1 fresh weight (FW) and 1.03 mg g−1 FW, respectively. The expressions of the studied candidate genes were significantly up-regulated in response to elicitation. Enhancement of capsaicinoids biosynthesis by elicitation with chitosan was found to be associated with the up-regulation of the expression of all the studied candidate genes. The present study shows the association of different enhancement potential of different elicitors with differential expression pattern of the candidate genes of capsaicinoids biosynthesis pathways in cell suspension cultures of C. chinense and the information provided may help in understanding the key mechanism for enhancement of capsaicinoids biosynthesis by elicitation. The present study reports the change in gene expression profiles of capsaicinoid biosynthesis pathway genes in response to elicitation in cell suspension cultures of Capsicum chinense Jacq.

Unique method for human villous trophoblasts isolation from placental tissue explants

Isolation of cytotrophoblasts from primary placental tissue may be costly and time consuming with variable results. In this paper, we provide a simple, affordable, and efficient method that may performed using common laboratory supplies to achieve consistent in vitro isolation of cytotrophoblasts from villous tissue. Trophoblast populations are identified based on morphology and phenotyping, which employs the timely extraction of villous nodes from the placenta prior to cultivation and isolation of nodal outgrowth by visual guidance for selective capture of cytotrophoblast populations and subculture. This method allows for the isolation of cytotrophoblasts free of contamination with other placental cell types. Isolated cells stain positive for the specific cytotrophoblast biomarker cytokeratin 7 and Human Chorionic Gonadotropin (HCG). Subcultured cells grow to confluency to establish monolayers that may be passaged in culture and later used to develop primary syncytiotrophoblasts over time. These primary cytotrophoblast populations may be employed using in in vitro placenta-on-a chip models to better understand placental cell biology and function, as well as physiological responses after exposure to toxicants, and infectious agents. This technique may be modified for selective isolation of specific cell types within different tissues from multiple organ systems.

Abstract 150: Obese Hypertensive Pregnant Rats Have Reduced Placental Growth Factor Whereas Increasing It's Levels Decreases Their Blood Pressure

While epidemiological studies have repeatedly shown that overweight and obesity are associated with hypertension during pregnancy, the mechanisms linking these co-morbidities are not as explored. Reports show reduced levels of the anti-hypertensive and pregnancy-related factor, placental growth factor (PlGF), in obese hypertensive pregnant humans. We tested the hypothesis that obese hypertensive pregnant rats have reduced PlGF whereas increasing its levels reduces their blood pressure. We utilized a genetic model of obesity in rats having heterozygous deficiency of the melanocortin-4 receptor (MC4R-def) compared to wild-type lean controls. By gestational day 19, body mass (341±8 vs 311±7 g), periaortic adipose tissue (10.9±1.1 vs 8.2±0.5 mg), retroperitoneal adipose tissue (4.4±0.4 vs 2.5±0.2 g), circulating levels of leptin (10±1 vs 6±1 ng/mL), and mean arterial blood pressure (116±2 vs 105±2 mmHg) were greater (all P&lt;0.05) in obese (N=17) compared to lean (N=14) pregnant rats. PlGF levels were lower (P&lt;0.05) in serum (61±6 vs. 78±7 pg/mL) and secreted from placental (58±3 vs 71±3 pg/mg) and periaortic adipose tissue (7±1 vs 15±2 pg/mL) explants in obese compared to lean groups. PlGF secretion from retroperitoneal adipose tissue was similar between obese (78±10 pg/mL) and lean (62±12 pg/mL) groups. Vascular expression of the PlGF receptor, vascular endothelial growth factor receptor 1 (VEGFR1), was lower (P&lt;0.05) in aortic tissue (832±60 vs 1106±91 pg/mg) but higher (P&lt;0.05) in small mesenteric arteries (214±23 vs. 151±17 pg/mg) from the obese pregnant rats. Chronic administration of recombinant human (rh)PlGF (180 ug/kg per day) from gestational day 13-19 produced detectable levels of human PlGF in serum from both lean (33±4 pg/mL) and obese (43±4 pg/mL) pregnant rats and reduced (P&lt;0.05) blood pressure in obese (102±3 mmHg) but not in lean (101±3 mmHg) groups. The treatment did not alter maternal body or fat masses or circulating leptin. In summary, PlGF is reduced in MC4R-def obese hypertensive pregnant rats, similar to findings in obese pregnant humans, whereas increasing the levels of PlGF reduced their hypertensive phenotype. Thus, treatment with rhPlGF may prove beneficial in attenuating blood pressure in obese hypertensive pregnancies.

Analysis of capsaicinoid biosynthesis pathway genes expression in callus cultures of Capsicum chinense Jacq. cv. ‘Umorok’

The placental tissue of the highly pungent chilli cultivar, Capsicum chinense Jacq. cv. ‘Umorok’, is used as explants for callus induction. Callus cultures were subcultured after every 32 days and growth curves for a period of six consecutive growth cycles were studied till a stable capsaicinoids producing callus cultures were obtained. The capsaicinoids content in placental tissue explants decreased gradually during the first 2 months of culture as the explants dedifferentiated to form friable callus while the biomass and capsaicinoid content did not show much change in the subsequent growth cycles. The maximum callus biomass of 7.8 g freshweight (FW) or 0.56 g dry weight (DW) per culture were obtained on the 24th day of every growth cycle and the maximum average capsaicinoids content (1.6 mg g−1 FW capsaicin and 0.78 mg g−1 FW dihydrocapsaicin) were obtained on the 20th day of every growth cycle. To investigate the underlying dynamics for capsaicinoid biosynthesis during callus formation, comparative gene expression analysis of the genes involved in capsaicinoid biosynthesis pathway were also studied by qRT-PCR analysis. When compared with placental tissue, all the studied genes showed reduced expression during callus formation, especially putative aminotransferase (pAMT) and pungent gene 1 (Pun1), which were extensively down regulated from the 3rd month onwards in the callus cultures. Therefore, the present study revealed that the down-regulated expression of mainly two putative genes in capsaicinoid biosynthetic pathway (pAMT and Pun1) resulted in lower accumulation of capsaicinoids in callus cultures compared to placental tissues of fruits. The present study reports the gene expression analysis of the genes involved in capsaicinoid biosynthesis pathway during callus formation from placental tissue explants of Capsicum chinense Jacq.

Zika virus infection in human placental tissue explants is enhanced in the presence of dengue virus antibodies in-vitro

The current Zika virus (ZIKV) outbreak is associated with neurological malformations and disorders in neonates. Areas of increased incidence of malformations may overlap with dengue-hyperendemic areas. ZIKV infection is enhanced by antibodies against dengue virus (DENV) in cell culture and inbred mice. Sufficiently powered clinical studies or primate studies addressing the enhancement of fetal ZIKV infection after previous dengue infection are not available. The human placenta is susceptible to ZIKV in vitro, but it is unknown whether antibody-dependent enhancement of ZIKV infection occurs at the placental barrier. Here we studied ZIKV infection in placental tissue in the presence of DENV-immune sera. Explants from the amniochorionic membrane, the chorionic villi, and the maternal decidua were infected with ZIKV in the presence of DENV type 1-, 2-, or 4-immune sera, or controls. Presence of DENV antibodies of any type enhanced the percentage of successful infections of organ explants between 1.42- and 2.67-fold, and led to a faster replication as well as significantly increased virus production. No enhancement was seen with yellow fever or chikungunya virus control sera. Pre-existing DENV antibodies may pose an increased risk of trans-placental ZIKV transmission.

Abstract P308: Differential VEGF-Dependent Induction of sFlt-1 From Vascular Endothelial and Placental Cells

Preeclampsia is a disorder of pregnancy characterized by new-onset hypertension and maternal endothelial dysfunction. Although the origins of the disease are unclear, it is believed that placental hypoxia/ischemia leads to the release of soluble factors which cause the maternal syndrome. The best characterized factor elevated in PE is soluble fms-like Tyrosine Kinase-1 (sFlt-1), a soluble splice variant of the VEGFR1 receptor which acts as a decoy receptor; and thus a VEGFA antagonist. Overproduction of this protein is believed to be a major contributor to the maternal endothelial dysfunction, causing much speculation that VEGF supplementation could be an effective supplementation could be an effective therapy. Recently we described a VEGFA chimera therapy (ELP-VEGFA) which alleviated the hypertension associated with placental ischemia in a rodent model, but also led to a dramatic increase in maternal circulating sFlt-1. Here we tested the hypothesis that chimeric VEGF would significantly increase sFlt-1 production from placental tissue in vitro. Surprisingly, there was no effect of ELP-VEGF on sFlt-1 production in either BeWo placental trophoblasts or in rodent placental tissue explants at either the mRNA or protein level. Instead, we found that vascular endothelial cells (HUVECs) responded robustly to ELP-VEGFA at the protein (6593 ± 199 vs 14547 ± 364 pg/ml, p&lt;0.05) and mRNA (100 ± 2 vs 401 ± 3.6%, p&lt;0.05) levels. This effect was unique to ELP-VEGFA as ELP-based proteins based on the VEGF family members VEGFB or PlGF had no effect on sFlt-1 production from either placental or endothelial cells. Finally, we found that 24hr post-administration of ELP-VEGF, pregnant rats demonstrated a trend for increased sFlt-1 mRNA in aorta (83 ± 7 vs 225 ± 63 AU, p&lt;0.05) but no increase in placental tissue. These data suggest that therapeutic peptides based on either VEGFB or PlGF could have significantly improved safety profiles over those based on the VEGFA sequence. Furthermore, it raises questions about the origin of much sFlt-1 protein in preeclamptic patients.

Changes in mitochondrial respiration in the human placenta over gestation

IntroductionPlacental mitochondria are subjected to micro-environmental changes throughout gestation, in particular large variations in oxygen. How placental mitochondrial respiration adapts to changing oxygen concentrations remains unexplored. Additionally, placental tissue is often studied in culture; however, the effect of culture on placental mitochondria is unclear. Material and methodsPlacental tissue was obtained from first trimester and term (laboured and non-laboured) pregnancies, and selectively permeabilized to access mitochondria. Respirometry was used to compare respiration states and substrate use in mitochondria. Additionally, explants of placental tissue were cultured for four, 12, 24, 48, or 96 h and respiration measured. ResultsMitochondrial respiration decreased at 11 weeks compared to earlier gestations (p = 0.05–0.001), and mitochondrial content increased at 12–13 weeks compared to 7–10 weeks (p = 0.042). In term placentae, oxidative phosphorylation (OXPHOS) through mitochondrial complex IV (p < 0.001), the relative proportion of OXPHOS CI (p < 0.001), the total capacity of the respiratory system (p = 0.003), and mitochondrial content (p < 0.001) were higher compared to first trimester. Respiration was increased (p ≤ 0.006–0.001) in laboured compared to non-laboured placenta. After four hours of culture, respiration was depressed compared to fresh tissue from the same placenta and continued to decline with time in culture. Markers of apoptosis were increased, while markers of autophagy, mitochondrial biogenesis, and mitochondrial membrane potential were decreased after four hours of culture. DiscussionRespiration and mitochondrial content alter over gestation/with labour. Decreased respiration at 11 weeks and increased mitochondrial content at 12–13 weeks may relate to onset of maternal blood flow, and increased respiration as a result of labour may be an adaptation to ischaemia-reperfusion. At term, mitochondria were more susceptible to changes in respiratory function relative to first trimester when cultured in vitro, perhaps reflecting changes in metabolic demands as gestation progresses. Metabolic plasticity of placental mitochondria has relevance to placenta-mediated diseases.

Isolation and characterization of trophoblasts from enzymatic explants of human term placenta.

In the present study, we describe a new method of isolation and culture of human villous and extravillous trophoblasts from term placenta. The cultivation of trypsinized placental villous tissue explants, followed by the isolation of cells from outgrowth islets allows for obtaining a cytotrophoblast subpopulation that is free from contamination by other cell types. Compared to other methods, our protocol is mild, simple and effective, does not request costly reagents and provides isolation of the mononuclear cytotrophoblast cell populations free from contamination by other types of placental cells. The isolated cells proliferated and formed a pleomorphic monolayer, where cells fused into a small number of binuclear or polynuclear syncytiotrophoblasts. Isolated cytotrophoblast cells expressed the specific epithelial intermediate filament cytokeratin 7 (CK7), the epithelium-specific cell-cell adhesion molecule E-cadherin and were CD9-, CD45- and vimentin-negative. Cyto- and syncytiotrophoblasts obtained by this method can be used as a model or tool for the fundamental research of differentiation and function of human placental cells, and can provide a new understanding of drug distribution in placenta. Their combination with other in vitro cell models can be useful for studying a variety of other aspects concerning placental functions, which will provide new knowledge for understanding immunology, endocrinology and development of placenta.

Ex vivo infection of human placental chorionic villi explants with Trypanosoma cruzi and Toxoplasma gondii induces different Toll-like receptor expression and cytokine/chemokine profiles.

Trypanosoma cruzi and Toxoplasma gondii present, respectively, low and high congenital transmission rates. The placenta as an immune regulatory organ expresses TLRs, leading to the secretion of cytokines. Both parasites are recognized by TLR-2, TLR-4, and TLR-9. Here, we studied if the parasites induce differences in TLR protein expression, cytokine profiles, and whether receptor inhibition is related to parasite infection. Placental tissue explants were infected ex vivo with each parasite, TLRs protein expression, cytokine profile and parasite infection were determined by Western blotting, ELISA and qPCR. Trypanosoma cruzi and Toxoplasma gondii infection is related to TLR-2 and TLR-4/TLR-9, respectively. Trypanosoma cruzi elicits an increase in TNF-α, IL-1β, IL-6, IL-8 and IL-10 cytokine secretion whereas T.gondii only increases the secretion of IL-8. The susceptibility of the placenta to each parasite is mediated partially by the innate immune response.

Effect of folic acid on human trophoblast health and function in vitro

IntroductionThe combined intake of folic acid (FA) from prenatal multivitamin supplements and fortified foods can result in FA intake values that exceed the tolerable upper intake level (UL). It is unclear what impact FA intake above the UL may have on the feto-placental unit. Our objective was to determine the effects of increasing concentrations of FA on trophoblast health and function in vitro. MethodsTwo human placental cell lines [HTR-8/SVneo (n = 5 experiments) and BeWo (n = 5 experiments)] and human placenta tissue explants (n = 6 experiments) were exposed to increasing concentrations of FA (2–2000 ng/mL) for 48-h. Intracellular total folate concentration, trophoblast proliferation, viability, apoptosis, placenta cell invasion and β-hCG hormone release were assessed. ResultsExposure to increasing FA concentrations resulted in higher intracellular total folate in placental cell lines and tissue explants (p < 0.05); yet, only minimal effects of excess folic acid were observed on the primary indicators of placental health and function studied. Specifically, treatment with excess folic acid (2000 ng/mL) resulted in reduced cellular viability in the villous trophoblast BeWo cell line and increased rates of proliferation in the HT8-8/SVneo extravillous trophoblast cell line (p < 0.05). Further, deficient concentrations of folic acid (2 ng/mL) resulted in decreased cell viability and invasive capabilities of the HTR-8/SVneo extravillous trophoblast cell line (p < 0.05). DiscussionOur results demonstrate that placental health and function may be compromised in conditions of folate deficiency, and not necessarily in conditions of excess FA. This finding supports the recommendation of prenatal folic acid supplementation in the North American population. Further work aimed at clarifying the therapeutic window of FA intake in the obstetrical population is warranted.

Influence of mitochondrial dynamics by selenium in trophoblasts

Influence of mitochondrial dynamics by selenium in trophoblasts

Leptin promotes HLA-G expression on placental trophoblasts via the MEK/Erk and PI3K signaling pathways

IntroductionThe development of the human haemochorial placenta requires complex regulatory mechanisms to protect invasive trophoblast cells from cytotoxic responses elicited by maternal immune cells. Leptin, the adipocyte derived hormone encoded by the Lep gene, is synthesized by placental trophoblasts and exerts pleiotropic effects on the immune system, including the promotion of inflammation and the activation of T cell responses. MethodsTo address its possible involvement in the modulation of maternal immune responses during pregnancy, we investigated the effect of leptin on the expression of the class Ib histocompatibility antigen HLA-G as one of the chief immunosuppressive strategies used by trophoblast cells. ResultsIn vitro incubation of the trophoblast derived Swan 71 and JEG-3 cell lines with 25–50 ng/ml recombinant leptin significantly boosted HLA-G mRNA and protein expression, and this effect was abrogated upon pharmacological inhibition of the PI3K-Akt and MEK-Erk signaling pathways. A similar stimulatory effect of leptin was observed in term placental tissue explants, though 10-fold higher doses were required for stimulation. Further, JEG-3 cells treated with a leptin antisense oligodeoxynucleotide displayed decreased HLA-G expression levels, which were partially recovered by addition of stimulating doses of exogenous hormone. Immunofluorescence and qPCR analysis confirmed leptin biosynthesis in placental tissue, further showing that invasive extravillous trophoblast cells were a main source of this hormone during the first trimester of normal pregnancies. DiscussionTaken together, our results show that leptin acts as an autocrine/paracrine signal promoting HLA-G expression in placental trophoblasts suggesting an important role in the regulation of immune evasion mechanisms at the fetal maternal interface.

Hexachlorobenzene and pentachlorobenzene accumulation, metabolism and effect on steroid secretion and on CYP11A1 and CYP19 expression in cultured human placental tissue

Hexachlorobenzene and pentachlorobenzene accumulation and the effect on CYP1A1, SULT1A, COMT and steroid secretion in term placental tissue were determined. Explants of placental tissue were exposed to between 0.02 and 2ng/ml HCBz or PeCBz for 6–72h. Accumulation was measured by capillary gas chromatography and quadrupole mass spectrometry. CYP1A1, SULT1A, COMT activity and progesterone secretion were analysed by EIA. Protein expression was quantified by Western blot; 6% HCBz and 7% PeCBz were detected in the tissue. Fast induction of CYP1A1 activity and protein expression in the presence of HCBz were observed. HCBz increased, while PeCBz decreased COMT protein expression. The stimulatory effect of HCBz, and the inhibitory of PeCBz on progesterone secretion and CYP11A1 protein expression were noted. Later activation of CYP1A1, inhibition of COMT protein expression and progesterone secretion by PeCBz suggest greater exposure to PeCBz and pointing at PeCBz as the main factor responsible for the disruption of placental function.

The effect of coelomic fluid on the production of cytokines by the first trimester human placenta

ObjectiveTo investigate the modulatory effects of coelomic fluid (CF) on the production of tumour necrosis factor-alpha (TNFα) and its receptors, TNF-R1 and TNF-R2, interferon gamma (IFNγ) and interleukin (IL)-10 by placental villous explants cultured under physiological oxygen (O2) concentration. Study designIn vitro culture of placental villous explants at atmospheric and physiological (6%) O2 levels at varying concentrations of CF. Main outcome measuresConcentration of TNFα, TNF-R1, TNF-R2, IFNγ and IL-10 in culture medium and villous explant homogenates, measured using flowcytometric bead arrays. ResultsThe median level and interquartile range of cytokines and receptors present in CF were: TNFα 0.9 pg/ml (0.85, 0.95); IFNγ 5.38 pg/ml (4.96, 6.18); IL-10 1.59 pg/ml (1.45, 1.76); TNF-R1 6043.65 pg/ml (5961.39, 6187.35) and TNF-R2 3563.52 pg/ml (3390.26, 3635.19). The PO2 of CF was 48.74 mmHg (SEM 1.59), equivalent to 6% O2.Increasing doses of CF significantly (p < 0.05) increased the levels of TNFα, TNF-R1 and TNF-R2 in the culture medium at both O2 concentrations.TNF-R1 levels measured in placental homogenate increased up to 2-fold at both O2 concentrations, but were significantly lower (1.96 fold; p = 0.03) at 6% O2 compared to 20% O2. ConclusionsThe exocoelomic cavity in humans contains high levels of both soluble TNF-R1 and TNF-R2. The addition of CF to placental tissue explants in culture modulates the placental secretion of TNFα-receptors and TNFα at both physiological and atmospheric O2 tension resulting in a concentration much higher than that found in the CF. This may contribute to the maternal inflammatory response previously reported in normal early pregnancy.

Novel model of placental tissue explants infected by cytomegalovirus reveals different permissiveness in early and term placentae and inhibition of indoleamine 2,3-dioxygenase activity

Human cytomegalovirus (HCMV) is the most common cause of viral intrauterine infection. Placental infection suggests hematogenous spread and permissiveness may vary according to the age of pregnancy. We set up and investigate permissivity of early and term placenta to HCMV with an ex vivo model of placental histocultures and evaluate the activity profile of IDO. Fourteen first trimester placentae were obtained following elective abortion and twelve term placentae after elective caesarean section. Fresh placental chorionic villi were isolated, washed and distributed on collagen sponge gels after overnight incubation with the virus. The culture medium was collected and fresh medium renewed regularly. Histology and immunohistochemistry showed preserved villous integrity in cultured placental histocultures. Infection could be seen in tissue sections of both early and term placentae, although early placentae were more permissive. Indoleamine 2,3-dioxygenase (IDO) is highly expressed in the placenta and is known to prevent maternal immune rejection. Constitutive IDO activity was higher in early, compared to term placentae and HCMV infection inhibited IDO activity in early placentae. IFN-γ-induced IDO activity was suppressed by HCMV in both early and term placentae. Our work shows a novel method of placenta organ culture. Our findings suggest that HCMV infects early placentae more strongly than term placentae. Early placental dysfunction through the inhibition of IDO activity may reveal a possible mechanism for miscarriages.

Application of Human Placental Villous Tissue Explants to Study ABC Transporter Mediated Efflux of 2,4-Dinitrophenyl-S-Glutathione

The purpose of this study was to characterize the human term placental villous tissue explant culture model as a tool to study the formation and efflux of 1-chloro-2,4-dinitrobenzene (CDNB) conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) as a model system for phase II metabolism and ATP-binding cassette (ABC) transporter-mediated cellular efflux. Placental tissue samples were obtained after cesarean section following normal pregnancies (n=9). Cultured villous tissue was monitored up to 48 h to study the effect of time in culture on biochemical parameters, formation and efflux of DNP-SG in the absence or presence of ATPase inhibitor sodium orthovanadate and the protein expression of ABC transporters - multidrug resistance associated protein 2 (MRP2), P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and enzyme glutathione-S-transferase isoform P1-1 (GSTP1-1). Villous tissue structure, tissue viability and expression of BCRP, GSTP1-1 remained unchanged, while expression of MRP2, P-gp and total tissue glutathione decreased with time in culture. Tissue integrity was unchanged up to 24 h but declined at 48 h. However, DNP-SG formation, DNP-SG efflux, and the extent of inhibition of DNP-SG efflux by sodium orthovanadate showed only minor changes through 48 h. Sodium orthovanadate decreased DNP-SG efflux, consistent with inhibition of apical ABC transporters. The results support the use of the cultured human term placental villous tissue explants model to study coordinated function of GSTP1-1 and apical ABC transporters in the formation and efflux of the model substrate DNP-SG.

Can the biology of VEGF and haem oxygenases help solve pre-eclampsia?

Pre-eclampsia, a pregnancy-specific multi-organ syndrome characterized by widespread endothelial damage, is a new risk factor for cardiovascular disease. No therapies exist to prevent or treat this condition, even to achieve a modest improvement in pregnancy length or birth weight. Co-administration of soluble VEGFR-1 [VEGF (vascular endothelial growth factor) receptor-1; more commonly known as sFlt-1 (soluble Fms-like tyrosine kinase-1)] and sEng (soluble endoglin) to pregnant rats elicits severe pre-eclampsia-like symptoms. These two anti-angiogenic factors are increased dramatically prior to the clinical onset of pre-eclampsia and are quite possibly the 'final common pathway' responsible for the accompanying signs of hypertension and proteinuria as they can be reversed by VEGF administration in animal models. HO-1 (haem oxygenase-1), an anti-inflammatory enzyme, and its metabolite, CO (carbon monoxide), exert protective effects in several organs against oxidative stimuli. In a landmark publication, we showed that the HO-1 pathway inhibits sFlt-1 and sEng in cultured cells and human placental tissue explants. Both CO and NO (nitric oxide) promote vascular homoeostasis and vasodilatation, and activation of VEGFR-1 or VEGFR-2 induced eNOS (endothelial nitric oxide synthase) phosphorylation, NO release and HO-1 expression. Our studies established the HO-1/CO pathway as a negative regulator of cytokine-induced sFlt-1 and sEng release and eNOS as a positive regulator of VEGF-mediated vascular morphogenesis. These findings provide compelling evidence for a protective role of HO-1 in pregnancy and identify it as a target for the treatment of pre-eclampsia. Any agent that is known to up-regulate HO-1, such as statins, may have potential as a therapy. Any intervention achieving even a modest prolongation of pregnancy or amelioration of the condition could have a significant beneficial health impact worldwide.