Our results provide molecular evidence that aberrant global placental gene expression changes in preeclampsia may be due to reduced oxygenation and that these events can successfully be mimicked by in vivo and in vitro models of placental hypoxia.

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Pre-eclampsia is a serious disorder of human pregnancy, characterized by decreased utero-placental perfusion and increased trophoblast cell death. Presently, the mechanisms regulating trophoblast cell death in pre-eclampsia are not fully elucidated. Herein, we have identified a novel Mtd/Bok splice isoform (Mtd-P) resulting from exon-II skipping. Mtd-P expression was unique to early-onset severe pre-eclamptic placentae as assessed by quantitative real-time-PCR and immunoblotting. Mtd-P overexpression in cell lines (BeWo: cytotrophoblast-derived; and CHO: ovary-derived) resulted in increased apoptotic cell death as assessed by caspase-3 cleavage, internucleosomal DNA laddering and mitochondrial depolarization. Moreover, Mtd-P expression increased under conditions of low oxygenation/oxidative stress in human villous explants. Antisense knockdown of Mtd under conditions of oxidative stress resulted in decreased caspase-3 cleavage. We conclude that under conditions of reduced oxygenation/ oxidative stress, Mtd-P causes trophoblast cell death in preeclampsia and hence may contribute to the molecular events leading to the clinical manifestations of this disease.

The human placenta is a unique organ in terms of oxygenation as it undergoes a transition from a low to a more oxygenated environment. This physiological switch in oxygen tension is a prerequisite for proper placental development and involves the hypoxia inducible factor (HIF). HIF is stable and initiates gene transcription under hypoxia, whereas in normoxia, interaction with the von Hippel-Lindau tumor suppressor protein (VHL) leads to rapid degradation of the HIF1A subunit. The degradation requires formation of a multiprotein complex (VHLCBC) and hydroxylation of HIF1A proline residues via members of the egg-laying-defective nine (EGLN) family. Herein, we have investigated the regulatory mechanisms of HIF1A expression during human placental development. Expression of HIF1A and VHL was high at 7-9 wk of gestation, when oxygen tension is low, and decreased when placental oxygen tension increases (10-12 wk of gestation). During early placentation, HIF1A localized in cytotrophoblasts, while VHL was present in syncytiotrophoblasts. At 10-12 wk, VHL appeared in cytotrophoblast cells, which coincided with the disappearance of HIF1A. At the same time the association of VHL and Cullin 2 as well as ubiquitination of HIF1A was maximal. EGLN1, EGLN2, and EGLN3 were also temporally expressed in an oxygen-dependent fashion, with greatest mRNA expression at 10-12 wk of gestation. Inhibition of EGLN activity increased HIF1A stability in villous explants and stimulated transforming growth factor beta 3 (TGFB3) expression consistent with promoter analyses showing that HIF1A transactivates TGFB3. These data demonstrate that during placental development, HIF1A is regulated by temporal and spatial changes in expression and association of molecules forming the multi-protein VHLCBC complex as well as prolyl hydroxylase activities.

BackgroundThe pathogenesis of preeclampsia, a serious pregnancy disorder, is still elusive and its treatment empirical. Hypoxia Inducible Factor-1 (HIF-1) is crucial for placental development and early detection of aberrant regulatory mechanisms of HIF-1 could impact on the diagnosis and management of preeclampsia. HIF-1α stability is controlled by O2-sensing enzymes including prolyl hydroxylases (PHDs), Factor Inhibiting HIF (FIH), and E3 ligases Seven In Absentia Homologues (SIAHs). Here we investigated early- (E-PE) and late-onset (L-PE) human preeclamptic placentae and their ability to sense changes in oxygen tension occurring during normal placental development.Methods and FindingsExpression of PHD2, FIH and SIAHs were significantly down-regulated in E-PE compared to control and L-PE placentae, while HIF-1α levels were increased. PHD3 expression was increased due to decreased FIH levels as demonstrated by siRNA FIH knockdown experiments in trophoblastic JEG-3 cells. E-PE tissues had markedly diminished HIF-1α hydroxylation at proline residues 402 and 564 as assessed with monoclonal antibodies raised against hydroxylated HIF-1α P402 or P564, suggesting regulation by PHD2 and not PHD3. Culturing villous explants under varying oxygen tensions revealed that E-PE, but not L-PE, placentae were unable to regulate HIF-1α levels because PHD2, FIH and SIAHs did not sense a hypoxic environment.ConclusionDisruption of oxygen sensing in E-PE vs. L-PE and control placentae is the first molecular evidence of the existence of two distinct preeclamptic diseases and the unique molecular O2-sensing signature of E-PE placentae may be of diagnostic value when assessing high risk pregnancies and their severity.

Placental hypoxia is causally implicated in fetal growth restriction and preeclampsia, with both occurring more frequently at high altitude (>2700 m; HA). The nuclear transcription factor hypoxia-inducible factor (HIF) may facilitate placental oxygen transport at HA by increasing erythropoiesis and placental angiogenesis. We therefore investigated HIF expression and its regulatory mechanisms in placentas from normal pregnancies at high (3100 m), moderate (1600 m), and sea level (75 m) altitudes. Moderate-altitude and sea level placentas did not differ, but HIF-1alpha and the von Hippel-Lindau tumor suppressor protein were overexpressed in HA placentas. The ability of von Hippel-Lindau tumor suppressor protein to form the E3 ubiquitin protein ligase complex, required for HIF-1alpha degradation, was unaltered in HA placentas. mRNA for factor-inhibiting HIF, a negative modulator of HIF-1alpha transactivation, was increased, but protein levels were diminished. Elevated HIF-1alpha likely contributed to the significant increase we report in HIF-1alpha downstream target proteins, transforming growth factor beta3 in the placenta, and vascular endothelial growth factor and erythropoietin in the maternal circulation. These circulating markers and lowered birth to placental weight ratios correlated with increased HIF-1alpha, thereby linking molecular and systemic physiological data. The HA response to chronic hypoxia resembles preeclampsia in several aspects, illustrating the utility of the HA model in understanding placental pathologies.

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Considering that interleukin 10 (IL10), transforming growth factor beta1 (TGFB1), and interferon gamma (IFNG) are involved in the susceptibility of BeWo trophoblast cells to Toxoplasma gondii infection, the aim of the present study was to investigate the effector mechanisms triggered by these cytokines in the control of T. gondii in BeWo cells. For this purpose, infected/uninfected BeWo cells were treated with IL10, TGFB1 (50 ng/ml), and IFNG (20 or 100 ng/ml) in order to verify the phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT3, and Smad2, parasite intracellular proliferation, as well as the Th1/Th2/IL17A cytokine production. The treatment of BeWo cells with IL10 and TGFB1 favored T. gondii proliferation, and these findings were associated with STAT3 and Smad2 phosphorylation, respectively (P < 0.05). Also, these cytokine treatments were able to down-modulate TNF alpha (TNFA) and IL6 production (P < 0.05). Low concentration of IFNG was unable to control T. gondii infection but was able to trigger STAT1 phosphorylation and up-regulate IL6 and IL17A production; whereas a high concentration of IFNG was unable to activate STAT1 but down-modulated IL6 and TNFA and increased T. gondii proliferation (P < 0.05). IL10, TGFB1, and IFNG regulate a differential T. gondii proliferation in BeWo cells because they distinctly trigger intracellular signaling pathways and cytokine production, especially IL6 and TNFA. Our data open new windows to understand the mechanisms triggered by IL10, TGFB1, and IFNG at the maternal-fetal interface in the presence of T. gondii, contributing to recognizing the importance of these effector mechanisms involved in the vertical transmission of this parasite.