PMC

Expression profiling of purinergic molecules in human placentas revealed that CD73 was elevated in preeclampsia patients. (A) ADA activities in human placentas (NT: n=9, PE: n=12) or mouse placentas from the dams with elevated placental adenosine (n=5 per group) were determined using a spectrophotometric assay. (**P<0.01, ND: not detected). (B) mRNA expression levels of CD73 in human placentas. (n=10 per group), (*P<0.05 vs NT). (C) Representative images of CD73 immunostaining in human placentas from NT or PE. Scale bar, 200μm. (D) CD73 activities to convert AMP to adenosine in human placentas were determined by enzyme-based assay. (NT: n=8, PE: n=10), (*P<0.05).

Genetic deletion of ADORA2B prevents placental elevated adenosine-induced preeclamptic features. (A) mRNA expression levels of adenosine receptors in placentas from dams with elevated placental adenosine were detected by real-time RT-PCR. Each value was expressed as a fold induction relative to Ada^+/− placentas. (n=6 each), (**P<0.01 vs Ada^+/−). (B) Schema of mating strategy to generate Adora2b^−/− dams with elevated placental adenosine. (C) Levels of adenosine in placentas from Adora2b^−/− dams with elevated placental adenosine were determined on E18.5 by HPLC. (n=4 each group), (*P<0.05 vs Ada^+/−/Adora2b^−/−). (D and E) Blood pressure measured by the tail-cuff method (D) and proteinuria (E) measured by ELISA. (Dams with elevated placental Ado: n=9, Adora2b^−/− dams: n=8), (D: *P<0.05 vs Dams with elevated placental Ado at the same time point, E: *P<0.05 vs Dams with elevated placental Ado).

Preeclamptic features caused by elevated placental adenosine are prevented by genetically restoring ADA to placentas or by ADA-enzyme therapy. (A) Schema of mating strategy of placental rescued ADA-deficient mice (Ada^−/−/Pl-Tg⁺). (B) ADA enzymatic activity in tissues determined by ADA zymogram analysis. (C) Adenosine levels in placentas from dams with elevated placental adenosine with or without PEG-ADA treatment (5 units on E5.5 and E12.5 by retro-orbital sinus injection) and placental ADA-rescued dams were determined on E18.5 by HPLC. (n=8 each group), (*P<0.05, **P<0.01). (D and E) Blood pressure measured by the tail-cuff method (D) and proteinuria (E) measured by ELISA. (Dams with elevated placental Ado: n=9, PEG-ADA treatment: n=5, Placental rescued dams: n=8), (D: *P<0.05, **P<0.01 vs Dams with elevated placental Ado at the same time point, E: *P<0.05 vs Dams with elevated placental Ado).

Pregnant mice with placental excess adenosine present with impaired placental vasculature with increased Flt-1 gene expression and elevated sFlt-1 levels in the maternal circulation. (A) Representative image of fetuses and placentas on E18.5 from dams with elevated placental adenosine. (B) Vascular structure of placentas from dams with elevated placental adenosine was assessed by CD31 immunostaining on E18.5. Scale bar; 200μm. (C) CD31-positive vessel density was determined. The data was quantified by counting CD31-positive vessels obtained from every three center areas in the labyrinth zone of three different samples in each group. (**P<0.01 vs Ada^+/−) (D) Flt-1 mRNA expression in the placentas on E18.5 determined by real-time RT-PCR. (n=6 each), (**P<0.01 vs Ada^+/−). (E) Maternal circulating sFlt-1 levels on E18.5. (Control dams: n=6, Dams with elevated Ado: n=4), (*P<0.05 vs Control dams).

Importance of excess adenosine coupled with enhanced ADORA2B signaling in placentas of preeclampsia patients. (A) Adenosine levels in the placentas of preeclampsia patients (PE) or normotensive pregnant women (NT) were detected by HPLC. (NT: n=9, PE: n=11), (**P<0.01). (B) Levels of ADORA2B mRNA in human placentas determined by real-time RT-PCR. (n=10 per group), (*P<0.05). (C) Levels of ADORA2B protein in human placentas detected by immunoblotting. (D) FLT-1 mRNA expression levels in human villous explants determined by real-time RT-PCR. Human villous explants were pretreated with or without 10 nM MRS1754 for 30 min and then treated with 1μM NECA for 24 hours. (n=3), (*P<0.05 vs non-treatment, ^†P<0.05 vs NECA treatment only). (E and F) ADORA2B agonist treatment significantly induced FLT-1 mRNA (E) and sFlt-1 secretion (F) in the cultured human villous explants. Human villous explants were treated with 1μM BAY60-6583 for 24 hours. (n=3), (*P<0.05, **P<0.01).

Elevated CD73 underlies increased placental adenosine and subsequent disease development via ADORA2B activation in an autoantibody-induced mouse model of PE. (A) Adenosine levels in mouse placentas on E18.5 were detected by HPLC. (n=5–8 per group), (**P<0.01 vs WT+NT-IgG, ^††P<0.01 vs WT+PE-IgG). (B) CD73 activities in IgG-injected mouse placentas were determined by enzyme-based assay. (n=4 each), (*P<0.05 vs NT-IgG). (C) Increased expression of Cd73 mRNA in placentas from PE-IgG-injected dams. (NT-IgG; n=6, PE-IgG; n=7), (**P<0.01 vs NT-IgG). (D) mRNA expression levels of adenosine receptors in mouse placentas were determined by real-time RT-PCR (n=6 each), (*P<0.05). (E and F) Blood pressure measured by the tail-cuff method (E) and proteinuria (F) measured by ELISA. (n=7–11 per group), (E: *P<0.05, **P<0.01 vs WT+NT-IgG at the same time point, ^†P<0.05, ^††P<0.01 vs WT+PE-IgG at the same time point, F: *P<0.05 vs WT+NT-IgG, ^†P<0.05 vs WT+PE-IgG). (G) Maternal circulating sFlt-1 levels on E18.5 det. (n=5–7 per group), (**P<0.01 vs WT+NT-IgG, ^†P<0.05 vs WT+PE-IgG). (H) Working model: Elevated placental adenosine coupled with enhanced ADORA2B signaling as a novel pathogenic factor for PE.

Pregnant mice with elevated placental adenosine display spontaneously developed hallmark features of preeclampsia. (A) Schema of mating strategy to generate pregnant mice with elevated placental adenosine (Ado). (B) Levels of adenosine in mouse placentas obtained on E12.5 or E18.5 detected by HPLC. (n=4 on E12.5, n=8 on E18.5 each), (*P<0.05 vs Ada^+/− on E12.5, *P<0.05 vs other groups on E18.5). (C) No significance of aternal circulating adenosine levels on E18.5. (n=4 each). (D) Blood pressure measured by the tail-cuff method from non-pregnant state (E0) until postpartum day 5 (Post-D5). (Control dams: n=10, Dams with elevated placental Ado: n=9), (*P<0.05, **P<0.01 vs E0, ^†P<0.05 vs control dams at the same time point). (E) Intra carotid mean arterial blood pressure (MAP) measured on E18.5. (Control dams: n=3, Dams with elevated Ado: n=4), (*P<0.05 vs Control dams). (F) Urine protein (ratio of albumin/creatinine in urine) was measured by ELISA. (Control dams: n=9, Dams with elevated Ado: n=7), (**P<0.01 vs other time points). (G) Electron microscopy of glomeruli. Glomeruli from dams with elevated placental adenosine possessed capillaries with markedly decreased lumenal spaces, with endothelial cytoplasmic swelling and a moderate expansion of the mesangium by mesangial cells and matrix. The visceral epithelial cells showed focal foot process effacement.