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Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. [ABSTRACT FROM AUTHOR]

Background: Preeclampsia is a prevalent hypertensive disorder of pregnancy and a leading cause of maternal and neonatal morbidity and mortality worldwide. This pathogenic condition is speculated to be caused by placental abnormalities that contribute to the maternal syndrome. However, the specific factors and signaling pathways that lead to impaired placentas and maternal disease development remain elusive.Methods and Results: Using 2 independent animal models of preeclampsia (genetically engineered pregnant mice with elevated adenosine exclusively in placentas and a pathogenic autoantibody-induced preeclampsia mouse model), we demonstrated that chronically elevated placental adenosine was sufficient to induce hallmark features of preeclampsia, including hypertension, proteinuria, small fetuses, and impaired placental vasculature. Genetic and pharmacological approaches revealed that elevated placental adenosine coupled with excessive A₂B adenosine receptor (ADORA2B) signaling contributed to the development of these features of preeclampsia. Mechanistically, we provided both human and mouse evidence that elevated placental CD73 is a key enzyme causing increased placental adenosine, thereby contributing to preeclampsia.Conclusions: We determined that elevated placental adenosine signaling is a previously unrecognized pathogenic factor for preeclampsia. Moreover, our findings revealed the molecular basis underlying the elevation of placental adenosine and the detrimental role of excess placental adenosine in the pathophysiology of preeclampsia, and thereby, we highlight novel therapeutic targets. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a serious hypertensive disease of pregnancy associated with the presence of the major angiotensin receptor AT1-agnostic autoantibodies (AT1-AAs) that cause the defining PE features when introduced into pregnant mice. To understand AT1-AA-mediated receptor activation in PE, we explored the role of tissue transglutaminases (TG2), the most ubiquitous member of a group of enzymes modifying proteins by catalyzing the formation of ε-(γ-glutamyl)-lysine isopeptide bonds. TG2, a versatile protein that can also function as a G protein, is enriched in the syncytiotrophoblasts of placenta, the key organ in PE pathogenesis. Western blot analysis and IHC-IF double staining initially revealed that TG2 and transglutaminase (TG)-mediated isopeptide modification levels are increased approximately 2X in PE vs normotensive (NT) placental tissue (n=4 or 5, p<0.05). Next, we identified a similar increase in PE plasma TG activity (NT=3.1±0.5, mild PE=5.4±0.6, severe PE=9.1±0.7milliunit/ml, n=26-29, p<0.01), and the increased plasma TG activity is positively correlated with the key disease features including blood pressure and proteinuria (r2>0.5, p<0.01). We also found a higher level of AT1 receptor with isopeptide modification in PE placentas by immunoprecipitation and IF colocalization. A pathogenic role for TG2 in PE is suggested by in vivo experiments in which TG inhibitor cystamine, or siRNA-mediated TG2 knockdown, significantly attenuated AT1-AA-induced PE features in pregnant mice including hypertension (PE=160±6 vs PE+cystamine=133±3mmHg, p<0.01, n=6; PE+control siRNA=161±3 vs PE+TG2 siRNA=137±7mmHg, p<0.05, n=5-6), proteinuria (PE=133±21 vs PE+cystamine=58±12ng albumin/mg creatine, p<0.01, n=6; PE+control siRNA=102±51 vs PE+TG2 siRNA=26±6ng albumin/mg creatine, p<0.05, n=4), and placental isopeptide modification. In trophoblasts, AT1-AA stimulation enhanced the interaction between AT1 receptor and TG2, and resulted in increased AT1 receptor stabilization via isopeptide modification, which is further confirmed by mutagenesis studies. Taken together, our results not only identify a pathogenic role of TG2 in PE, but also shed light on a previously unrecognized mechanism of TG2-mediated GPCR stabilization. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a prevalent life-threatening complication of pregnancy featuring hypertension, placental abnormality, and renal damage. The condition is associated with the presence of agonistic autoantibodies (AT1-AA) capable of activating the major angiotensin receptor, AT1R, and believed to contribute to disease pathogenesis. Tissue transglutaminase (tTG), a prominent member of a family of enzymes that crosslink proteins by catalyzing the formation of inter- or intra-molecular ε-(γ-glutamyl)-lysine isopeptide bonds, is known to interact with G protein-coupled receptors and contribute to hypertension. Thus, we hypothesize that AT1-AA may function via tTG to crosslink AT1R and contribute to pathophyhsiology of PE. To test this hypothesis, using immunoprecipation with anti-tTG antibody, we successfully pulled down more AT1R from human trophoblast (HTR) cells treated with IgG from PE women than IgG from normotensive (NT) women, thereby providing initial evidence for the promotional effect of AT1-AA on tTG and AT1R interaction. Subsequently, we measured tTG and its modification on AT1R in both NT and PE placentas. We found increased levels of tissue transglutaminase (∼2 folds), ε-(γ-glutamyl)-lysine isopeptides (∼2 folds), and AT1R with ε-(γ-glutamyl)-lysine isopeptide bonds at the microvillous membrane of PE placentas, indicating that tTG-induced AT1 receptor modification is elevated in PE placenta. Finally, in an effort to determine the role of elevated tTG in AT1-AA-induced PE in vivo, we treated PE-IgG injected pregnant mice with cystamine, a potent tTG inhibitor. Compared with PE-IgG injected mice, less AT1 receptor with ε-(γ-glutamyl)-lysine isopeptide bonds was found in the placental labyrinth zone of PE-IgG injected mice treated with cystamine. Moreover, cystamine treatment significantly attenuated key features associated with PE including hypertension (from 159.5±5.6 to 132.6±2.7 mm Hg) and proteinuria (from 106.5±37.8 to 38.5±6.9 ng albumin/mg creatine) in the PE-IgG-injected pregnant mice as well. Taken together, here we report a previously unrecognized role of tTG in the pathogenesis of PE by crosslinking AT1Rs and suggest a novel therapeutics for the disease. [ABSTRACT FROM AUTHOR]

Background: High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia., Methods: Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m., Results: This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation., Conclusions: Together, human and mouse studies reveal novel mechanisms of hypoxia adaptation and potential therapeutic approaches for counteracting hypoxia-induced tissue damage., (© 2016 American Heart Association, Inc.)