Reduced methylation of the thromboxane synthase gene is correlated with its increased vascular expression in preeclampsia

Abstract

Preeclampsia is characterized by increased thromboxane and decreased prostacyclin levels, which predate symptoms, and can explain some of the clinical manifestations of preeclampsia, including hypertension and thrombosis. In this study, we examined DNA methylation of the promoter region of the thromboxane synthase gene (TBXAS1) and the expression of thromboxane synthase in systemic blood vessels of normal pregnant and preeclamptic women. Thromboxane synthase is responsible for the synthesis of thromboxane A(2), a potent vasoconstrictor and activator of platelets. We also examined the effect of experimentally induced DNA hypomethylation on the expression of thromboxane synthase in a neutrophil-like cell line (HL-60 cells) and in cultured vascular smooth muscle and endothelial cells. We found that DNA methylation of the TBXAS1 promoter was decreased and thromboxane synthase expression was increased in omental arteries of preeclamptic women as compared with normal pregnant women. Increased thromboxane synthase expression was observed in vascular smooth muscles cells, endothelial cells, and infiltrating neutrophils. Experimentally induced DNA hypomethylation only increased expression of thromboxane synthase in the neutrophil-like cell line, whereas tumor necrosis factor-α, a neutrophil product, increased its expression in cultured vascular smooth muscle cells. Our study suggests that epigenetic mechanisms and release of tumor necrosis factor-α by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in maternal systemic blood vessels, contributing to the hypertension and coagulation abnormalities associated with preeclampsia.

Figure 1

Boxplot of proportion methylated (β-values) in omental arteries by subject group for TBXAS1 gene as determined by the HumanMethylation27 BeadChip. Methylation was significantly lower in preeclamptic patients (n=7) than in normal pregnant patients (n=5) with no overlap between the groups. ***p = 0.00037, FDR = 0.042.

Figure 2

Representative sections for blood vessels in omental fat from normal pregnant and preeclamptic women immunostained for thromboxane synthase. A) There was no brown staining for thromboxane synthase in negative control sections. B) Blood vessels of normal pregnant women showed little or no staining for thromboxane synthase. C, D, E and F) Vessels of preeclamptic women showed significant brown staining for thromboxane synthase. Staining for thromboxane synthase in preeclamptic blood vessels was observed in endothelium (black arrows), vascular smooth muscle cells (red arrows), and leukocytes (blue arrows), which were either adhered to the endothelium or infiltrated into the wall of the vessel (F). A: adipocyte; VL: vessel lumen. Magnification and scale bar are shown on each image.

Figure 3

Results for immunohistochemical staining for thromboxane synthase in omental blood vessels from normal pregnant (NP) and preeclamptic (PE) women. A) Visual staining score for thromboxane synthase was significantly higher in blood vessels of preeclamptic women as compared to normal pregnant women. B) Optical density of staining for thromboxane synthase was also significantly higher in preeclamptic blood vessels and significantly correlated with the visual score (r=0.93). C) Preeclamptic women had significantly higher percentage of blood vessels stained for thromboxane synthase as compared to normal pregnant women. D) Preeclamptic women had significantly higher percentage of blood vessels with leukocyte stained for thromboxane synthase as compared to normal pregnant women. Data are presented as means ± SEM. ***p< 0.001.

Figure 4

Thromboxane synthase expression in omental arteries from normal pregnant and preeclamptic women. A) Fold change in gene expression of the TBXAS1 in omental fat arteries of normal pregnant (NP) and preeclampsia (PE) women. Gene expression of TBXAS1 was significantly increased in omental arteries from preeclamptic women (n=8) as compared to normal pregnant women (n=5). B) Representative Western blot for thromboxane synthase protein expression in omental arteries of normal pregnant and preeclamptic women. C) Density of thromboxane synthase plotted as percentage of average normal pregnant. Thromboxane synthase expression was significantly increased in arteries of preeclamptic women (n=6) as compared to normal pregnant women (n=4). Data are shown as means ± SEM. **p < 0.01.

Figure 5

Thromboxane synthase expression in HL-60 cells. A) Fold change in the expression for the TBXAS1 gene in HL-60 cells treated with PMA for 24 hours, 5-Aza for 48 hours or with 5-Aza for 48 hours and then with PMA for 24 hours (n=8). 5-Aza is an agent that inhibits DNA methylation when incorporated into DNA during cell division. The expression of TBXAS1 was significantly increased in 5-Aza treated cells as compared to controls. Activation with PMA after treatment with 5-Aza caused an even greater increase in TBXAS1 expression as compared to control, PMA alone or 5-Aza alone. B) Representative Western blot for thromboxane synthase expression in HL-60 cells. C) Density of thromboxane synthase plotted as percentage of control (n=5). Treatment with 5-Aza significantly increased thromboxane synthase expression as compared to control. Activation with PMA after treatment with 5-Aza resulted in significant increase in thromboxane synthase expression as compared to control, PMA alone and 5-Aza alone. Data are shown as means ± SEM. Groups with different letters are significantly different, p<0.001.

Figure 6

Thromboxane B₂ (TXB₂) secretion into the media by HL-60 cells treated with PMA for 24 hours, 5-Aza for 48 hours or with 5-Aza for 48 hours and then with PMA for 24 hours. TXB₂ secretion was significantly increased in 5-Aza or PMA treated cells as compared to controls. Combined treatment of PMA and 5-Aza caused an even greater increase in TXB₂ secretion as compared to control, PMA alone or 5-Aza alone. Data are shown as means ± SEM, n=6. Groups with different letters are significantly different, p<0.001.

Figure 7

Thromboxane synthase expression in human vascular smooth muscle cells treated with TNFα. A) Fold change in the expression for TBXAS1 gene in cultured VSMCs treated with TNFα for 48 hours. The expression of TBXAS1 was significantly increased in TNFα treated cells as compared to controls. B) Representative Western blot for thromboxane synthase protein expression in cultured VSMCs. C) Density of thromboxane synthase plotted as percentage of control. Treatment with TNFα significantly increased thromboxane synthase protein expression as compared to control. Data are shown as means ± SEM, n=6. ***p < 0.001, **p<0.01.