DNA methylation‐reprogrammed oxytocin receptor underlies insensitivity to oxytocin in pre‐eclamptic placental vasculature

Abstract Pre‐eclampsia is associated with inadequate placental blood flow and placental ischaemia. Placental vascular tone is essential for maintaining adequate placental blood flow. Oxytocin is increased in placental system at late pregnancy and onset of labour, and presented strongly concentration‐dependent contractions in placental vascular, suggesting that oxytocin could be involved in regulating placental vascular tone and circulation. However, information about the reactivity of oxytocin in pre‐eclamptic placental vasculature is limited. This study used a large number of human placentas to reveal the pathophysiological changes and its underlying mechanisms of oxytocin‐induced vasoconstrictions in placental vessels under pre‐eclamptic condition. Present study found that oxytocin‐induced contractions were significantly decreased in human pre‐eclamptic placental vasculature, associated with a deactivated transcription of oxytocin receptor gene. The deactivated oxytocin receptor gene transcription was ascribed to a relatively higher DNA methylation status of CpG islands in oxytocin receptor gene promoter. This study was first to reveal that a hyper‐methylation of CpG islands in oxytocin receptor gene promoter, leading to a relatively low pattern of oxytocin receptor expression, was responsible for the decreased sensitivity of oxytocin in pre‐eclamptic placental vessels.

maternal and foetal circulation systems; (b) immuno-protection and releasing of chemicals necessary for pregnancy progress. It is undeniable that, as a new foeto-maternal vascular organ, the maintenance of placental functions is dependent on sufficient placental perfusion and adequate blood flow via placental circulatory system. 6 Generally, this system includes a placental vascular tree (composed of chorionic plate arteries and their branches, terminal villi and veins). 6 Vascular tone in this system is essential for maintaining adequate placental blood flow. Because placental vessels lack autonomic innervation, 7 local vasoconstrictors and vasodilators are of paramount importance for controlling blood flow in placental circulation. Abnormalities in constriction and relaxation of the placental vasculature will affect blood flow in the placenta, contributing to placental ischaemia and an abnormal course of pregnancy such as PE. 8,9 Therefore, the abnormal vascular reactivity of endogenous vaso-activators in pre-eclamptic placental vasculature offers interesting model for investigations of mechanisms as well as targets of treatments for PE. However, so far, there has been limited information on the abnormal vascular reactivity of endogenous vaso-activators such as oxytocin (OXT) in pre-eclamptic placental vasculature.
Oxytocin is a nine-amino acid neuropeptide that is known to play a critical role in foetal expulsion and breastfeeding, and has been recently implicated in mammalian social behaviour. 10,11 The actions of both central and peripheral OXT are mediated through oxytocin receptor (OXTR), which is encoded by a single gene. In the context of pregnancy, OXT and OXTR are rich in the human placenta. 10,12 There is an increase in OXT concentrations in maternal plasma and the placenta with the onset of labour. 10,11 As a classical vasoconstrictor, OXT is predominantly known as a potent stimulator of uterine smooth muscle, and studies on isolated placental vessels have suggested an important role for this compound in the maintenance of placental vascular tone and circulation. 13 However, information about the reactivity of OXT in pre-eclamptic placental vasculature is limited. This was the first study using a large number of human placental vessels, to reveal the pathophysiological changes and its underlying mechanisms of OXT-induced vasoconstrictions in placental vessels under the pre-eclamptic condition, which would provide new information for better understanding the pre-eclamptic placental vascular dysfunctions and placental ischaemia. Pre-eclamptic pregnancies were defined by the onset of hypertension during pregnancy (blood pressure ≥ 140/90 mm Hg, with no prior hypertension history) and consistent proteinuria (≥300 mg/d). 1,14 The clinical characteristics of all participants were summarized in Table 1.
Human placental vessels (HPV) (HPV-A1/A2, first-, second-order branch of blood vessels in the placenta, mainly the main stem villous arteries; HPV-A3, branch of the main stem villous arteries with diameter around 100 m) were carefully isolated. HPV were cut into rings approximately 3-5 mm in length and mounted in a multi-myograph system for recording of isometric tension (PowerLab 16/SP and Chart 5). 15 Vessel rings were adjusted to maintain a suitable passive force and equilibrated for 60 minutes. Potassium chloride (KCl, 120 mmol/L) was used to achieve optimal resting tension before adding drugs. The contractions elicited by KCl were a reference for contractile capacity in response to drugs. 16 The vessel rings were contracted with cumulative concentrations of OXT (10 −10 to 10 −4 mol/L in HPV-A1/A2; 10 −8 to 10 −4 in HPV-A3). Atosiban (10 −5 mol/L) was used for pre-treating segments for 30-60 minutes before application of OXT. All drugs were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

| Quantitative real-time PCR and Western blot analysis
Total RNA was isolated from placental vessels using Trizol reagent, and was reversed transcribed using the first-strand cDNA Synthesis ∆∆Ct method was used to comparatively quantify the abundance of mRNA levels. The qRT-PCR primer sequences were listed in Table 3.
The protein abundance of OXTR in placental vessels was assessed by Western blot analyses normalized to β-actin. Antibodies were purchased from Sigma-Aldrich or CST (Cell Signaling Technology, Inc China). All experiments were repeated three times with independently prepared tissue, and performed as previously described. 19

| Data analysis and statistics
Data are presented as mean ± SEM, and analysed by a two-tailed

| Oxytocin-induced contractions in human placental vessels
In both HPV-A1/A2 and HPV-A3, there were no significant differences in KCl-induced maximal contractions between normal NP and PE group ( Figure 1B and D). Whereas, in both of HPV-A1/A2 and HPV-A3 from PE group, OXT-induced contractions were significantly decreased with a reduced maximal response and pD2 (−log [50% effective concentration]) as compared to NP group ( Figure 1A, C and E, P < 0.05). These data indicated that pre-eclamptic placental vessels were significantly insensitive to OXT.

| Expression of OXTR in human placental vessels
To investigate whether the decreased OXT-mediated vasoconstrictions were correlated with the altered OXTR expression in pre-eclamptic placental vessels, OXTR mRNA and protein levels were determined. As shown in Figure 2A-C, there was a significant decrease in protein of OXTR in both of HPV-A1/A2 and HPV-A3 from PE group.
To determine whether OXTR expression was regulated through transcriptional mechanism, we measured the mRNA abundance of OXTR. Figure 2B-D showed that compared with NP group, the mRNA levels of OXTR in HPV-A1/A2 and HPV-A3 were decreased in PE group. These data suggested that the decreased sensitivity to OXT in pre-eclamptic placental vessels was correlated with the down-regulated OXTR, and the deactivated transcription of OXTR gene.

| The effect of atosiban on OXT-induced contractions in human placental vessels
To further determine whether the decreased OXTR was associated with OXT-induced vasoconstrictions in PE group, vascular rings were pre-treated with atosiban (OXTR-specific antagonist). As shown in Figure 3, atosiban almost completely blocked OXT-mediated contractions in both of HPV-A1/A2 and HPV-A3, without differences between NP and PE groups after pre-treated with atosiban, demonstrating that the decreased OXT-mediated vasoconstrictions in PE group were due to the down-regulated OXTR.

| DNA methylation of CpG locus at OXTR gene promoter in human placental vessels
The expression of a gene may be down-regulated through an increase in DNA methylation of its promoter, CpG islands. 18 TA B L E 3 The primers used in this study    Tables 2   and 4. Correlation analysis between OXTR gene methylation and expression was also conducted. As shown in Figure 4F, there was a significantly inverse correlation between the methylation status of 22 CpG sites in OXTR gene promoter and OXTR gene expression.

| D ISCUSS I ON
This study found that OXT-induced vascular contractions were significantly decreased in pre-eclamptic placental vasculature. The decreased sensitivity to OXT in pre-eclamptic vessels was correlated with the down-regulated OXTR, as well as the deactivated transcription of OXTR gene. OXTR gene transcription was linked with DNA methylation status of CpG islands in OXTR gene promoter. To the best of our knowledge, this is the first study to reveal that a hypermethylation of certain CpG islands in OXTR gene promoter led to a relatively reduced pattern of OXTR expression, which was responsible for the decreased sensitivity to OXT in placental vessels under pre-eclamptic conditions. Thus, the data not only offered new information for further understanding the pathological features and mechanisms of PE, but also underlined a role of epigenetic-mediated gene expression in pre-eclamptic placental vascular dysfunctions such as placental ischaemia.
In uterus, large (HPV-A1/A2) and small (HPV-A3) vascular branches in the placenta play a key role in maintaining a balance of placental circulation and provide adequate blood flow for development of foetus. Present data showed that in both pre-eclamptic large and small placental vessels, OXT-induced vascular contractions were significantly decreased with a reduced maximal response and pD2 as compared to normal pregnancy, indicating that pre-eclamptic placental vessels were significantly insensitive to OXT. To date, information about the reactivity of OXT in pre-eclamptic placental vessels was limited. One study in 1989 reported that OXT produced weak contractions in placental vessels, and no significantly differences in OXT-induced contractions between normal and pre-eclamptic placental vessels. 20 Based on these results, previous study suggested that the effects of OXT on placental vascular tone in human term pregnancy are negligible. However, considering huge variation in human participants and the sample size was small (only six per group), it is hard to confirm OXT effects on human placental vessels. The similar situations were noted regarding vascular effects of acetylcholine on placental vessels recently, as obvious controversy results existed before. 8,16,21,22 To solve the problem, we significantly increased sample size in order to reach repeatable results. 15,16 In the present study, the large number of human samples (60 independent vessel tests, more than 40 placentas per group) was used for functional analysis. At least 85% in 60 independent tests showed OXT-induced contractions were significantly decreased in pre-eclamptic placental vessels.
Oxytocin exerts its effects on cells through interaction with OXTR, a G-protein coupled receptor that upon ligand binding transduces signal to the nucleus. To investigate whether OXTR mediates OXT-decreased vasoconstrictions in pre-eclamptic placental vessels, placental vessel rings were pre-treated with atosiban (an OXTR-specific antagonist). Atosiban almost completely inhibited OXT-induced contractions in placental vessels, without significant differences in OXT-induced contractions between normal and preeclamptic placental vessels after pre-treatment with atosiban. These results indicated that OXTR mediated OXT-decreased vasoconstrictions in pre-eclamptic placental vessels. Furthermore, the protein levels of OXTR were decreased in pre-eclamptic placental vessels.
Interestingly, the mRNA abundance of OXTR was also significantly decreased in pre-eclamptic placental vessels. These data demonstrated that the decreased OXT-mediated vasoconstrictions in preeclamptic placental vessels were correlated with the deactivated transcription of OXTR.
Numerous studies reported that transcription of OXTR was found to be partly regulated by epigenetic mechanisms in animal and human tissues. [23][24][25][26][27] DNA methylation is a major epigenetic mechanism that regulates gene transcription and there is a strong correlation between gene expression and DNA methylation in gene promoter. 18 Hyper-methylation of CpG sites in the promoter of genes is often associated with suppression of transcription. 18  The present study found a special pattern of OXT-mediated vascular contractions in pre-eclamptic placental vasculature.
The decreased sensitivity to OXT in pre-eclamptic vessels was correlated with the down-regulated OXTR and deactivated transcription of OXTR, which was linked to a hyper-methylation of

CO N FLI C T O F I NTE R E S T
The authors have none to declare.