HIF1 inhibitor acriflavine rescues early-onset preeclampsia phenotype in mice lacking placental prolyl hydroxylase domain protein 2.

Preeclampsia is a serious pregnancy disorder that lacks effective treatments other than delivery. Improper sensing of oxygen changes during placentation by prolyl hydroxylases (PHDs), specifically PHD2, causes placental hypoxia-inducible factor-1 (HIF1) buildup and abnormal downstream signaling in early-onset preeclampsia, yet therapeutic targeting of HIF1 has never been attempted. Here we generated a conditional (placenta-specific) knockout of Phd2 in mice (Phd2-/- cKO) to reproduce HIF1 excess and to assess anti-HIF therapy. Conditional deletion of Phd2 in the junctional zone during pregnancy increased placental HIF1 content, resulting in abnormal placentation, impaired remodeling of the uterine spiral arteries, and fetal growth restriction. Pregnant dams developed new-onset hypertension at midgestation (E9.5) in addition to proteinuria and renal and cardiac pathology, hallmarks of severe preeclampsia in humans. Daily injection of acriflavine, a small molecule inhibitor of HIF1, to pregnant Phd2-/- cKO mice from E7.5 (prior to hypertension) or E10.5 (after hypertension had been established) to E14.5 corrected placental dysmorphologies and improved fetal growth. Moreover, it reduced maternal blood pressure and reverted renal and myocardial pathology. Thus, therapeutic targeting of the HIF pathway may improve placental development and function, as well as maternal and fetal health, in preeclampsia.

Dichotomy in hypoxia-induced mitochondrial fission in placental mesenchymal cells during development and preeclampsia: consequences for trophoblast mitochondrial homeostasis.

Dynamic changes in physiologic oxygen are required for proper placenta development; yet, when low-oxygen levels persist, placental development is halted, culminating in preeclampsia (PE), a serious complication of pregnancy. Considering mitochondria's function is intimately linked to oxygen changes, we investigated the impact of oxygen on mitochondrial dynamics in placental mesenchymal stromal cells (pMSCs) that are vital for proper placental development. Transmission electron microscopy, proximity ligation assays for mitochondrial VDAC1 and endoplasmic reticulum IP3R, and immunoanalyses of p-DRP1 and OPA1, demonstrate that low-oxygen conditions in early 1st trimester and PE promote mitochondrial fission in pMSCs. Increased mitochondrial fission of mesenchymal cells was confirmed in whole PE placental tissue sections. Inhibition of DRP1 oligomerization with MDiVi-1 shows that low oxygen-induced mitochondrial fission is a direct consequence of DRP1 activation, likely via HIF1. Mitophagy, a downstream event prompted by mitochondrial fission, is a prominent outcome in PE, but not 1st trimester pMSCs. We also investigated whether mesenchymal-epithelial interactions affect mitochondrial dynamics of trophoblasts in PE placentae. Exposure of trophoblastic JEG3 cells to exosomes of preeclamptic pMSCs caused heightened mitochondrial fission in the cells via a sphingomyelin-dependent mechanism that was restored by MDiVi-1. Our data uncovered dichotomous regulation of mitochondrial fission and health in human placental mesenchymal cells under physiologic and pathologic hypoxic conditions and its impact on neighboring trophoblast cells.

Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction.

Aberrant ceramide build-up in preeclampsia, a serious disorder of pregnancy, causes exuberant autophagy-mediated trophoblast cell death. The significance of ceramide accumulation for lysosomal biogenesis in preeclampsia is unknown. Here we report that lysosome formation is markedly increased in trophoblast cells of early-onset preeclamptic placentae, in particular in syncytiotrophoblasts. This is accompanied by augmented levels of transcription factor EB (TFEB). In vitro and in vivo experiments demonstrate that ceramide increases TFEB expression and nuclear translocation and induces lysosomal formation and exocytosis. Further, we show that TFEB directly regulates the expression of lysosomal sphingomyelin phosphodiesterase (L-SMPD1) that degrades sphingomyelin to ceramide. In early-onset preeclampsia, ceramide-induced lysosomal exocytosis carries L-SMPD1 to the apical membrane of the syncytial epithelium, resulting in ceramide accumulation in lipid rafts and release of active L-SMPD1 via ceramide-enriched exosomes into the maternal circulation. The SMPD1-containing exosomes promote endothelial activation and impair endothelial tubule formation in vitro. Both exosome-induced processes are attenuated by SMPD1 inhibitors. These findings suggest that ceramide-induced lysosomal biogenesis and exocytosis in preeclamptic placentae contributes to maternal endothelial dysfunction, characteristic of this pathology.

JMJD6 Dysfunction Due to Iron Deficiency in Preeclampsia Disrupts Fibronectin Homeostasis Resulting in Diminished Trophoblast Migration.

The mechanisms contributing to excessive fibronectin in preeclampsia, a pregnancy-related disorder, remain unknown. Herein, we investigated the role of JMJD6, an O2- and Fe2+-dependent enzyme, in mediating placental fibronectin processing and function. MALDI-TOF identified fibronectin as a novel target of JMJD6-mediated lysyl hydroxylation, preceding fibronectin glycosylation, deposition, and degradation. In preeclamptic placentae, fibronectin accumulated primarily in lysosomes of the mesenchyme. Using primary placental mesenchymal cells (pMSCs), we found that fibronectin fibril formation and turnover were markedly impeded in preeclamptic pMSCs, partly due to impaired lysosomal degradation. JMJD6 knockdown in control pMSCs recapitulated the preeclamptic FN phenotype. Importantly, preeclamptic pMSCs had less total and labile Fe2+ and Hinokitiol treatment rescued fibronectin assembly and promoted lysosomal degradation. Time-lapse imaging demonstrated that defective ECM deposition by preeclamptic pMSCs impeded HTR-8/SVneo cell migration, which was rescued upon Hinokitiol exposure. Our findings reveal new Fe2+-dependent mechanisms controlling fibronectin homeostasis/function in the placenta that go awry in preeclampsia.

Increased placental mitochondrial fusion in gestational diabetes mellitus: an adaptive mechanism to optimize feto-placental metabolic homeostasis?

INTRODUCTION: Gestational diabetes mellitus (GDM), a common pregnancy disorder, increases the risk of fetal overgrowth and later metabolic morbidity in the offspring. The placenta likely mediates these sequelae, but the exact mechanisms remain elusive. Mitochondrial dynamics refers to the joining and division of these organelles, in attempts to maintain cellular homeostasis in stress conditions or alterations in oxygen and fuel availability. These remodeling processes are critical to optimize mitochondrial function, and their disturbances characterize diabetes and obesity. METHODS AND RESULTS: Herein we show that placental mitochondrial dynamics are tilted toward fusion in GDM, as evidenced by transmission electron microscopy and changes in the expression of key mechanochemical enzymes such as OPA1 and active phosphorylated DRP1. In vitro experiments using choriocarcinoma JEG-3 cells demonstrated that increased exposure to insulin, which typifies GDM, promotes mitochondrial fusion. As placental ceramide induces mitochondrial fission in pre-eclampsia, we also examined ceramide content in GDM and control placentae and observed a reduction in placental ceramide enrichment in GDM, likely due to an insulin-dependent increase in ceramide-degrading ASAH1 expression. CONCLUSIONS: Placental mitochondrial fusion is enhanced in GDM, possibly as a compensatory response to maternal and fetal metabolic derangements. Alterations in placental insulin exposure and sphingolipid metabolism are among potential contributing factors. Overall, our results suggest that GDM has profound impacts on placental mitochondrial dynamics and metabolism, with plausible implications for the short-term and long-term health of the offspring.

Factor inhibiting HIF1-A novel target of SUMOylation in the human placenta.

Adaptations to changes in oxygen are critical to ensure proper placental development, and impairments in oxygen sensing mechanisms characterize placental pathologies such as preeclampsia. In this study, we examined the involvement of SUMOylation, a reversible posttranslational modification, in the regulation of the asparaginyl hydroxylase Factor Inhibiting Hypoxia Inducible Factor 1 (FIH1) in the human placenta in development and in disease status. FIH1 protein abundance and spatial distribution in the developing placenta directly correlated with oxygen tension in vivo. Immunofluorescence analysis showed that early on FIH1 primarily localized to nuclei of cytotrophoblast cells, while after 10 weeks of gestation it was present in nuclei and cytoplasm of both cytotrophoblast and syncytiotrophoblast cells. Exposure of choriocarcinoma JEG-3 cells to hypoxia induced FIH1 SUMOylation by promoting its association to SUMO2/3. Transfection of JEG-3 cells with FIH1 constructs containing SUMO-mutated sites revealed that SUMOylation of FIH1 by SUMO2/3 targeted it for proteasomal degradation, particularly in hypoxia. SUMOylation of FIH1 directly impacted on HIF1A activity as determined by HIF-responsive luciferase assay. Co-immunoprecipitation analyses revealed enhanced FIH1-SUMO2/3 associations early in development, when FIH1 levels are low, while deSUMOylation of FIH1 by SENP3 increased later in gestation, when FIH1 levels are rising. In preeclampsia, decreased FIH1 protein expression associated with impaired deSUMOylation by SENP3 and increased association with the ubiquitin ligase RNF4. We propose a novel mode of regulation of FIH1 stability by dynamic SUMOylation and deSUMOylation in the human placenta in response to changing oxygen tension, thereby mediating HIF1A transcriptional activity in physiological and pathological conditions.

Dynamic regulation of HIF1Α stability by SUMO2/3 and SENP3 in the human placenta.

INTRODUCTION: Hypoxia-inducible factor 1A (HIF1A) stability is tightly regulated by hydroxylation and ubiquitination. Emerging evidence indicates that HIF1A is also controlled by the interplay between SUMO-specific ligases, which execute protein SUMOylation, and Sentrin/SUMO-specific proteases that de-SUMOylate target proteins. Given the significance of HIF1A in the human placenta, we investigated whether placental HIF1A is subject to SUMOylation in physiological and pathological conditions. METHODS: Placentae were obtained from normal and pregnancies complicated by preeclampsia. Human choriocarcinoma JEG3 cells were maintained at either 21% or 3% oxygen or exposed to sodium nitroprusside (SNP). Cells were transfected with SUMO2/3 constructs with and without Mg132, a proteasome inhibitor. Expression, distribution and associations of SUMO/SENPs and HIF1A were evaluated by Western blotting, immunohistochemistry and co-immunoprecipitation. RESULTS: HIF1A-SUMO2/3 associations peaked at 9-10 weeks, while its deSUMOylation by SENP3 was greatest at 10-12 weeks. In E-PE, HIF1A deSUMOylation by SENP3 was significantly elevated, while HIF1A-SUMO2/3 associations remained constant. In vitro, overexpression of SUMO2/3 de-stabilized HIF1A in hypoxia, and abrogated HIF1A expression following Mg132 treatment in normoxia. Hypoxia and SNP treatments promoted SENP3 nuclear redistribution from nucleoli to the nucleoplasm where it associates with HIF1A. CONCLUSION: During early placental development, SUMOylation events control HIF1A stability in an oxygen-dependent manner. In E-PE, enhanced deSUMOylation of HIF1A by SENP3 may in part contribute to increased HIF1A activity and stability found in this pathology.

Aberrant TGFß Signalling Contributes to Dysregulation of Sphingolipid Metabolism in Intrauterine Growth Restriction.

CONTEXT: Sphingolipids function as key bioactive mediators that regulate cell fate events in a variety of systems. Disruptions in sphingolipid metabolism characterize several human pathologies. OBJECTIVE: In the present study we examined sphingolipid metabolism in intrauterine growth restriction (IUGR), a severe disorder complicating 4-7% of pregnancies at increased risk of perinatal morbidity and mortality, which is characterized by placental dysfunction and augmented trophoblast cell death rates. DESIGN, SETTING, AND PARTICIPANTS: Placentae from early severe IUGR with documented abnormal umbilical artery Doppler defined as absence or reverse of end diastolic velocity and a birth weight below the fifth percentile for gestational age were collected (n = 58). Placental tissues obtained from healthy, age-matched preterm and term deliveries (n = 46; TC, n=28) were included as controls. RESULTS: Sphingolipid analysis by tandem mass spectrometry revealed elevated sphingosine and decreased ceramide levels in placentae from pregnancies complicated by IUGR relative to age-matched controls. Sphingosine accumulation was due to accelerated ceramide breakdown via increased acid ceramidase (ASAH1) expression/activity caused by augmented TGFß signalling via the ALK5/SMAD2 pathway. In addition, a marked reduction in sphingosine kinase 1 (SPHK1) expression/activity due to impaired TGFß signalling via ALK1/SMAD1 contributed to the sphingosine buildup in IUGR. Of clinical significance, ALK/SMAD signalling pathways were differentially altered in IUGR placentae. CONCLUSIONS: Altered TGFß signalling in IUGR placentae causes dysregulation of sphingolipid metabolism, which may contribute to the increased trophoblast cell death typical of this pathology.