Acute Hypoxia and Chronic Ischemia Induce Differential Total Changes in Placental Epigenetic Modifications

Abstract

Preeclampsia is a common obstetrical complication, hallmarked by new-onset hypertension. Believed to result from placental insufficiency and chronic placental ischemia, the symptoms of preeclampsia are caused by release of pathogenic factors from the placenta itself, although the mechanisms of their regulation are in many cases unknown. One potential mechanism is through changes in placental epigenetic chromatin modifications, particularly histone acetylation and DNA methylation. Here, we determined the effects of chronic ischemia on global epigenetic modifications in the rodent placenta in vivo and acute hypoxia in BeWo placental trophoblast cells in vitro. Placental insufficiency via uterine artery restriction increased maternal blood pressure and fetal demise while decreasing placental and fetal mass. Global placental histone H3 acetylation levels were significantly decreased at H3 K9, K14, K18, K27, and K56. Interestingly, when BeWo-immortalized placental trophoblast cells were cultured in oxygen concentrations mimicking healthy and ischemic placentas, there was a significant increase in acetylated at K9, K18, K27, and K56. This was associated with a small but significant decrease in placental acetyl-CoA, suggesting depletion in the source of acetyl group donors. Finally, while global methylation of cytosine from placental DNA was low in both groups of animals (<1%), there was ∼50% increase in 5-mC in response to chronic ischemia. This suggests acute hypoxia and chronic ischemia induce differential global changes in histone acetylation in the placenta and that chronically altered metabolic profiles could affect histone acetylation in the placenta, thereby regulating production of pathogenic factors from the placenta during preeclampsia.

Keywords: DNA methylation; epigenetics; histones; preeclampsia.

Figure 1.

BeWo trophoblast cells increase histone H3 acetylation acutely in response to hypoxia in vitro. After 24 hours culture in oxygen concentration mimicking healthy and hypoxic placentas (8% and 1% oxygen), lysates were analyzed for H3 acetylation (1F). In response to hypoxic treatment, there was significantly (P < .05) increased acetylation of H3K9 (∼175%)(1A), H3K14 (∼164%)(1B), H3K18 (∼176%)(1C), and H3K56 (∼166%)(1E), with a strong trend for increased acetylation of H3K27 (∼141%)(1D) when compared to controls. n = 8 per group. Average pup and placental weights were determined for each dam, and averages compared for each. Statistical comparisons with corresponding P values are indicated by connecting lines.

Figure 2.

Placental ischemia induces significant increases in maternal blood pressure (A) and fetal demise (D), significant decreases in placental mass (B), and a trend for a decrease in viable fetal mass (C). N = 8 per group. Statistical comparisons with corresponding P values are indicated by connecting lines.

Figure 3.

Histone H3 acetylation is altered in response to chronic placental ischemia. Whole placental lysates from Sham-operated or RUPP-treated rats were analyzed for global levels of histone H3 acetylation by Western blotting (3F). In response to chronic ischemia, placental histone H3 had significantly lower acetylation at H3K9 (∼66%)(3A), H3K14 (∼66%)(3B), H3K18 (60%)(3C), H3K27 (∼76%)(3D), and H3K56 (∼68%)(3E) when compared to control. N = 8 per group. Statistical comparisons with corresponding P values are indicated by connecting lines. NP indicates normal pregnant (sham), RUPP indicates reduced uterine perfusion pressure.

Figure 4.

Placental acetyl-CoA is significantly decreased in chronic placental ischemia. Acetyl-CoA was extracted from placental tissue and assayed by flourometric assay. In response to reduced placental perfusion, there was a significant decrease (∼15%, P < .05) in the tissue level of acetyl-CoA in whole placental lysates. N = 8 per group. CoA indicates coenzyme A.

Figure 5.

Global 5-Me-cytosine is increased in response to chronic placental ischemia. Whole placentas from sham-operated and RUPP-treated rats were examined for global cytosine methylation by ELISA. In response to placental ischemia, global methylation levels were increased by approximately 2-fold. N = 8 per group. Statistical comparisons with corresponding P values are indicated by connecting lines. RUPP indicates reduced uterine perfusion pressure; ELISA, enzyme-linked immunosorbent assay.