Expression of AT1R, AT2R and AT4R and Their Roles in Extravillous Trophoblast Invasion in the Human

Abstract

The placental renin-angiotensin system (RAS) is active from early pregnancy and may have a role in placentation. Angiotensin II (AngII) acts via binding to receptor types AT1R and AT2R. Recently smaller peptide members of the angiotensin family have been recognised as having biological relevance. Angiotensin (3-8) (AngIV) has a specific receptor (AT4R) and evokes hypertrophy, vasodilatation and vascular inflammatory response. The aim of this study was to characterise placental expression of AT1R, AT2R and AT4R, and to determine whether AngII and AngIV regulate extravillous trophoblast (EVT) invasion, apoptosis and proliferation. Placental samples were obtained from women undergoing elective surgical termination of pregnancy (TOP) at 8–10 weeks gestation (early TOP), 12–14 weeks gestation (mid TOP) or at delivery following normal pregnancy or with pre-eclampsia (PE). Immunohistochemistry and qRT-PCR were performed to determine placental mRNA and protein expression of AT1R, AT2R and AT4R at all gestational ages. EVT invasion following culture with AngII or AngIV was assessed in early placental tissue using Matrigel invasion assays. Invasion was assessed on day 6 of culture and placental explants were harvested for immunohistochemical analysis of apoptosis and proliferation. The results from qRT-PCR and immunohistochemistry showed placental AT1R expression which did not vary with gestation. The highest levels of expression of AT2R were found in early and mid TOP placentae compared to term pregnancy. Expression of AT4R was increased in term placentae, with a significant reduction in PE placentae. Moreover, culture with AngIV or AngII increased EVT invasion from placental explants, which showed increased trophoblast proliferation and reduced apoptosis. This study has characterised expression of AT4R and AT1R and AT2R in human placenta throughout normal pregnancy and in PE. Both AngIV and AngII may play an important role in normal pregnancy.

Introduction

The circulating renin-angiotensin system (RAS) is important for regulation of blood pressure and electrolyte balance. Until recently AngII was thought to be the major bioactive peptide, however recently it has emerged that the shorter peptides, produced by aminopeptidase mediated cleavage of AngII, including angiotensin (3-8) (AngIV), also have roles in regulating cardiovascular function. There are two major angiotensin receptors, angiotensin II receptor 1 (AT1R) and angiotensin II receptor 2 (AT2R), which have similar binding affinities for AngII [1]. AngII mediates most of its effects via binding with AT1R, ultimately triggering vasoconstriction, proliferation, angiogenesis or inflammation [1]. AT2R is predominantly expressed in fetal tissues [2], and AngII binding increases apoptosis, causes vasodilation and is involved in fetal tissue development [3].

AngIV appears to mediate various effects in different tissues via binding with high affinity to its specific receptor, the AT4R [4]. AngIV can also bind to AT1R and AT2R with low affinity. The active site of AT4R has been identified to be an insulin-regulated aminopeptidase (IRAP) [5], also known as cysteine aminopeptidase, oxytocinase or placental leucine aminopeptidase (P-LAP). AT4R expression has been localised in both endothelial [6] and smooth muscle cells [7] indicating physiological roles in regulating blood flow. AngIV can increase blood flow via a mechanism mediated by AT4R and nitric oxide [8]. Due to its localization in extravillous trophoblast (EVT) Ino et al. [9] suggested that this receptor may be involved in regulating the invasion of EVT during placentation.

In addition to the circulating RAS, tissue based systems are found in heart, brain and reproductive tissues. The fetal–maternal interface comprises both the fetal placental tissue RAS and the maternal decidual tissue RAS. Factors involved in stimulating decidualisation, including oestrogen and progesterone, have been identified as playing a role in regulating the local RAS, which is thought to be important in spiral artery remodelling.

Plasma renin concentration and activity, as well as AngII levels are increased in pregnancy, but vascular responsiveness to AngII is decreased [10]. In contrast, pre-eclamptic patients exhibit exaggerated pressor responses to AngII, although circulating concentrations are lower compared to control pregnancies [10]. Although the underlying mechanism remains to be elucidated, there is growing evidence to indicate that dysregulation of both the tissue based and circulating RAS may be involved in the pathophysiology of PE [10].

The intervillous space is exposed to altering oxygen gradients during the first 10–12 weeks of gestation [11] during which time EVT invades through the decidua. Following this period of EVT invasion into uterine tissues, placental oxygen tension rapidly increases due to the maternal perfusion of the villous tips [12]. This occurs episodically, and is predicted to have effects similar to those seen in hypoxia/reperfusion, leading to the development of local placental oxidative stress. In the normal early placenta, antioxidants protect the placenta from undue damage due to oxidative stress [11].

The exact mechanism underlying the development of PE remains unknown, but it has been suggested that shallow trophoblast invasion resulting in inadequate spiral artery transformation may underlie its pathogenesis [13]. Increased mitochondrial generation of reactive oxygen species (ROS) and synthesis through xanthine oxidase (XO) [14] and NADPH oxidase result in increased levels of ROS in PE and this is combined with decreased expression of antioxidants [15]. Locally-generated AngII is a potent stimulus to NADPH oxidase secretion [16]. NADPH oxidase is composed of a number of subunits, the catalytic machinery of the enzyme being provided by gp91^phox. A family of genes homologous to gp91^phox known as Nox 1-5 has been discovered; Nox 4 is expressed in fetal tissues, placenta and vascular cells [17].

The objectives of the current study were two-fold. The first was to examine the placental expression of AT1R, AT2R and AT4R, relate this to the expression of NADPH oxidase and XO throughout normal pregnancy and compare the expression with that in PE. The second was to investigate a potential role for these receptors in normal placental development.