Increased Arterial Responsiveness to Angiotensin II in Mice Conceived by Assisted Reproductive Technologies

Abstract

Since the first report in 1978, the number of individuals conceived by Assisted Reproductive Technologies (ART) has grown incessantly. In parallel, with the recent emergence of possible underlying mechanisms of ART-induced epigenetic changes in the renin-angiotensin system, the cardiovascular repercussions of ART in mice and human offspring (including arterial hypertension, vascular dysfunction, and cardiac remodeling) have become increasingly recognized. Here, we hypothesized that ART may increase arterial responsiveness to angiotensin II (ANG II) by epigenetically modifying the expression of its receptors. To test this hypothesis, we assessed the vasoconstrictor responsiveness to ANG II in isolated aortas from ART and control mice. We also examined ANG II receptor (ATR) type 1 and 2 expression and the promoter methylation of the At1aR, At1bR and At2R genes. We found that the vasoconstrictor response to ANG II was markedly increased in ART mice compared to controls. This exaggerated vasoconstrictor responsiveness in ART mice correlated with a significant increase in the ANG II receptor (ATR) type 1 to ATR type 2 protein expression ratio in the aorta; this was mainly driven by an increase in AT1R expression, and by hypomethylation of two CpG sites located in the At1bR gene promoter leading to increased transcription of the gene. We conclude that in mice, ART increase the vasoconstrictor response to ANG II in the aorta by epigenetically causing an imbalance between the expression of vasoconstrictor (AT1R) and vasodilator (AT2R) ANG II receptors. Unbalanced expression of AT1R and AT2R receptors seems to be a novel mechanism contributing to ART-induced arterial hypertension in mice.

Keywords: DNA methylation; angiotensin II receptors; assisted reproductive technologies; hypertension.

Figure 1

Vasoconstrictor response of aortic rings to ANG II in presence of L-NNA, measured with wire myography and expressed in % of maximal KCl contraction (n ≥ 6/group). Values are the Means ± SEM. Data were analyzed by two-way ANOVA. ** p < 0.01, ART vs. Ctrl.

Figure 2

(A) AT1R and AT2R protein expression and (B) AT1R/AT2R ratio in aorta measured by Western blotting (n ≥ 7/group). (C) A representative western blot image. Values are mean ± SE. * p < 0.05, ART vs. Ctrl. ns = not significant.

Figure 3

(A) At1bR gene promoter DNA methylation level determined by bisulfite pyrosequencing. (B) ATRs mRNA quantification in aorta by RT-qPCR in (n ≥ 10/group). Values are mean ± SE. * p < 0.05, ART vs. Ctrl. ns = not significant.