Human placental adenosine receptor expression is elevated in preeclampsia and hypoxia increases expression of the A2A receptor

Abstract

Placental hypoxia as a result of impaired trophoblast invasion is suggested to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine, and the actions of adenosine are mediated through four adenosine receptors, A(1), A(2A), A(2B) and A(3). We investigated the presence, distribution and expression of adenosine receptor subtypes in the human placenta, the expression of the adenosine receptors in placentas from pregnancies complicated by preeclampsia, small for gestational age (SGA) infants and uncomplicated pregnancies, and the effect of hypoxia on placental adenosine receptor expression. Immunofluorescent microscopy localized A(1), A(2A), A(2B) and A(3) adenosine receptors to the syncytiotrophoblast, endothelial cells and myofibroblasts within the human placenta. Adenosine receptor protein and message expression levels were significantly higher in placentas from preeclamptic pregnancies with or without SGA infants, but not different in pregnancies with SGA infants alone. In vitro exposure of placental villous explants to hypoxia (2% oxygen) increased the expression of A(2A) adenosine receptor 50%. These data indicate that all four known adenosine receptors are expressed in the human placenta and adenosine receptor expression is significantly higher in pregnancies complicated by preeclampsia. These data are consistent with the hypothesis that differences in placental adenosine receptors may contribute to alterations in placental function in preeclampsia.

Figure 1

Localization of A₁, A_2A, A_2B and A₃ receptors in human placenta. Immunohistochemistry of frozen sections was performed as described in Materials and Methods. A, D, G, J) Positive fluorescent staining with an A₁, A_2A, A_2B and A₃(A-268, A-269, sc7505, A4229) adenosine receptor antibody. B) Fluorescent staining of endothelial cells with an CD31 antibody (BD 50389), E, H) of syncytiotrophoblast with cytokeratin 18 antibody (M7010) and K) of fibroblasts and myofibroblasts with an α-smooth muscle actin antibody (ab7817). Co-localization of adenosine receptors on endothelial (C), syncytiotrophoblast (F, I) or fibroblast cells (L), merged photographs. Arrows represent adenosine receptors only, arrowheads show co-localization areas. Scale bars: 20μm.

Figure 2

Adenosine receptor A₁, A_2A, A_2B and A₃ protein expression in placental biopsies of women with uncomplicated (NP), preeclamptic (PE), preeclamptic with SGA (PE & SGA) and SGA pregnancies (N=6 for each group). A) Representative western Blot of A₁, A_2A, A_2B and A₃ receptors and β-actin as loading control. B) Placentas of women with PE and PE & SGA have a higher protein expression of adenosine receptors A₁, A_2A, A_2B and A₃. Scatter plots represent fold changes compared to uncomplicated pregnancies (dashed line) after normalization to β-actin. A dashed line indicates a ratio of 1.0 and a solid line indicates the median fold change of each group. All P values refer to comparisons of data from uncomplicated pregnancies.

Figure 3

Adenosine receptor A₁, A_2A, A_2B and A₃ mRNA expression in placentas of women with preeclamptic (PE), preeclamptic with SGA (PE & SGA) and SGA pregnancies compared to women with uncomplicated pregnancies (NP), (dashed line, N=6 for each group). Placentas of women with PE and PE & SGA have a higher mRNA expression of adenosine receptors A₁, A_2A, A_2B and A₃ compared to uncomplicated pregnancies. SGA pregnancies are not different from uncomplicated pregnancies. Scatter plots represent fold changes compared to uncomplicated pregnancies (dashed line). A dashed line indicates a ratio of 1.0 and a solid line indicates the median fold change for each group. All P values refer to comparisons of data from uncomplicated pregnancies.

Figure 4

Adenosine receptor A_2A and HIF-1α expression in placental villous explants of women with uncomplicated (NP) and preeclamptic (PE) pregnancies after incubation for 24 h at 21% and 2% oxygen (N=6 for each group). A) Representative western blot of adenosine receptor A_2A, HIF-1α and β-actin. B) Adenosine receptor A_2A protein expression is significantly higher after exposure to hypoxia in placental villous explants of women with uncomplicated pregnancies. Values were normalized to β-actin. C) Real time RT-PCR revealed that adenosine receptor A_2A mRNA expression is significantly higher after exposure to hypoxia in placental villous explants of women with uncomplicated pregnancies. Scatter plots represent fold changes compared to 21% oxygen of NP or PE pregnancies. D) Hypoxia induced HIF-1α proteins in placental villous explants from uncomplicated and preeclamptic pregnancies as assessed by western analyses. A dashed line indicates a ratio of and a solid line the median fold change of each group. All P values refer to comparisons of data from normoxia.