Ontogeny of Placental Structural Development and Expression of the Renin–Angiotensin System and 11β-HSD2 Genes in the Rabbit

Placenta 30 (2009) 590–598

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Ontogeny of Placental Structural Development and Expression of the Renin–Angiotensin System and 11b-HSD2 Genes in the Rabbit A.M. McArdle a, K.M. Denton a, D. Maduwegedera a, K. Moritz b, R.L. Flower a, C.T. Roberts c, * a

Department of Physiology, Monash University, Melbourne, Australia School of Biomedical Sciences, University of Queensland, Brisbane, Australia c Discipline of Obstetrics and Gynaecology, University of Adelaide, Adelaide, South Australia 5005, Australia b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 14 April 2009

Common pregnancy complications are associated with impaired placental development. This study aimed to characterise the ontogeny of structural correlates of rabbit placental function, its expression of genes encoding components of the renin–angiotensin system (RAS), as well as 11b-hydroxysteroid dehydrogenase type 2 (11b-HSD2) mRNA since these are known to be expressed by the placenta and are associated with pregnancy complications, including preeclampsia and intrauterine programming. Placentae were collected at gestational age (GA) 14, 21 and 28 (term ¼ 32 days). Gene expression was analysed using real time PCR and placental structures were quantified via image analyses. The volume densities and volumes of trophoblast, fetal capillaries, maternal blood space, surface density and surface area of trophoblast all progressively increased, while the arithmetic mean barrier thickness of trophoblast decreased across gestation. Maternal plasma renin activity (PRA) was positively correlated with volumes of trophoblast and maternal blood space, surface density and surface area of trophoblast. Placental renin mRNA declined (Y62%; P < 0.01) across gestation and was negatively correlated with maternal PRA (GA0), fetal and placental weights, placental angiotensin type 1 and 2 receptors (AT1R and AT2R) mRNA and volume of trophoblast. AT1R mRNA expression was increased by 92% (P < 0.001) across gestation. AT2R mRNA expression was w81% (P < 0.01) greater at GA14 compared to GA21. Placental 11bHSD2 mRNA expression was w74% greater (P < 0.01) at GA21 than GA14, but by GA28 was similar to that at GA14. These data show that changes in placental gene expression are associated with key events in placental and fetal development, indicating that the rabbit provides a good model for investigations of pregnancy perturbations that alter the RAS or programme the fetus. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Pregnancy Placenta Renin–angiotensin system Rabbit

1. Introduction The placenta is a highly specialised organ that plays an essential role in fetal growth and development and in maternal adaptation to pregnancy [1,2]. Common pregnancy complications are associated with poor placental development, including inadequate trophoblast invasion and altered angiogenesis [3]. In addition, the quality of the maternal adaptation to pregnancy is also known to contribute to pregnancy success. Current evidence suggests that alterations to essential nutritional and endocrine functions of the placenta may occur in response to changes in the maternal environment [2]. The placenta forms the interface between the fetus and the mother. Therefore changes in the maternal environment may alter placental development and

* Corresponding author. Tel.: þ61 8 83033118; fax: þ61 8 83034099. E-mail address: [email protected] (C.T. Roberts). 0143-4004/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2009.04.006

function [2]. These placental changes may further contribute to an adverse intrauterine milieu and increase the risk of the development of adult diseases, including cardiovascular disease in offspring, a phenomenon known as intrauterine programming [2,4]. Good animal models of placental differentiation and growth are important to enable investigation into the causes and mechanisms involved in intrauterine programming and aberrant maternal adaptation to pregnancy. We have previously shown in a rabbit model of chronic maternal hypertension that offspring have increased arterial pressure as adults [5]. The rabbit as a model for the study of reproductive function and development has many advantages. The uterus is duplex [6] allowing controlled independent experiments in each uterine horn. Rabbit placentation is similar to that of humans as both have invasive discoid hemochorial placentas, although the rabbit placenta is hemodichorial while that of the human placental is hemomonochorial [7]. However the pattern of feto-maternal interdigitation at the placental exchange

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surface is the primary difference between the two species. The human placental interface is villous in nature, whereas in the rabbit a labyrinth is formed [7–9]. Throughout gestation the placenta undergoes specific periods of growth and remodelling in order to provide the nutrients and hormones necessary for fetal wellbeing while maintaining maternal health [9,10]. Therefore disruptions to placental development, including changes to vasculogenesis and angiogenesis, and alterations in trophoblast expression of transporters, enzymes and hormones, are associated with alterations in placental function [10]. Defective placental differentiation and function is likely to be both a cause and consequence of maternal hypertensive complications such as pre-existing hypertension and preeclampsia (PE) [11]. In approximately one third of pregnancies with these complications, intrauterine growth restriction (IUGR) is also an important feature that increases morbidity and mortality in offspring [12]. Placental invasion, differentiation and expansion of functional capacity across gestation is highly orchestrated both spatially and temporally [1]. A remarkably complex interaction of placental and maternal growth factors, cytokines, receptors, hormones and proteins is involved. During normal pregnancy the renin–angiotensin system (RAS) is activated. However, in essential hypertension the level of RAS activation is inappropriate for the given level of arterial pressure. Similarly in PE occurring in previously normotensive women, both the local placental and systemic RAS are aberrant [13]. Thus differential regulation of the RAS may contribute to the complications associated with hypertensive pregnancies. The local placental RAS acts in an autocrine/paracrine way and is thought to be involved in increasing vascular permeability, angiogenesis and decidualisation during implantation and placental morphogenesis [14]. All of the components of the RAS have been found in the human utero-placental unit [15], as well as at various stages throughout pregnancy in other species including rabbit [15], rat [16] and guinea pig [15]. Although the levels of RAS gene and protein expression have been shown to differ between species, this is thought to reflect species specific variations in placental structure [14]. The enzyme 11b-HSD2, catalyses the reaction of active cortisol into its inactive metabolites, thus regulating fetal exposure to maternal glucocorticoids [17]. Placental 11b-HSD2 has been shown to be important in the regulation of fetal growth, with reductions in placental 11b-HSD2 associated with human IUGR and intrauterine programming of adult disease [18]. Furthermore, 11b-HSD2 has been shown to be regulated by angiotensin II, the effector molecule in the RAS cascade [19]. Due to the importance of normal placental structural development and function to the growth and development of the fetus, this study aimed to characterise the ontogeny of structural correlates of placental function, including trophoblast differentiation and placental vascularisation. In addition, herein we report the mRNA expression of components of placental RAS, namely renin, angiotensin type 1 (AT1R) and type 2 (AT2R) receptors, as well as 11b-HSD2 across gestation, as the ontogenic expression of these factors has not been previously described in the rabbit. It Table 1 Maternal measurements at gestational age (GA) 0, 14, 21 and 28. GA0 (n ¼ 23)

GA14 (n ¼ 8)

GA21 (n ¼ 7)

GA28 (n ¼ 8)

Body weight (kg) 3.15  0.09 a 3.14  0.17 a 3.67  0.16 b 3.60  0.13ab Mean arterial 75.92  1.01 ab 72.8  1.09 ab 80.64  3.03 a 70.06  2.64 b pressure (mmHg) Plasma renin activity 2.47  0.25 a 1.46  0.34 a 2.38  0.81 a 6.72  1.57 b (ng Ang/ml/h) Values expressed as mean  SEM. P < 0.05 for all dissimilar letters.

Fig. 1. Fetal weight (g) (A), placental weight (g) (B) and fetal-to-placental weight ratio (FW:PW) (C) at gestational age (GA) 14, 21 and 28. Values expressed as estimated marginal mean  SEM. P < 0.05 for all dissimilar letters.

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Fig. 2. Representative Masson’s trichrome stained placental section showing giant cells (light blue staining) and labyrinth (purple staining) at gestational age 21. Photographed with 10 objective lens.

was hypothesised that placental differentiation and the ontogenic expression of components of the RAS and 11b-HSD2 mRNA in the rabbit would be related and comparable to those seen in mammals with similar discoid hemochorial placentation, including humans. Thus we aimed to demonstrate whether the rabbit may provide a good model for investigating hypertensive complications of pregnancy and in particular, whether the expressions of components of the placental RAS are similar between rabbits and humans. 2. Methods 2.1. Animals Nulliparous female English cross-bred rabbits (15  1 wk of age, 2.8  0.1 kg) were housed individually with temperature maintained between 20 and 22  C and a 12 h light-dark cycle. Rabbits were fed a meal of 100 g of high-fibre low starch rabbit pellets (Glen Forrest Stockfeeders, WA, Australia) and water was provided ad libitum. All experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Monash University Animal Ethics Committee. 2.2. Maternal measurements Maternal body weight, mean arterial pressure (MAP) and plasma renin activity (PRA) were measured in conscious rabbits prior to mating and at the time of tissue collection (gestational age (GA) 14, 21 or 28; mothers n ¼ 8, n ¼ 7, n ¼ 8, respectively; term ¼ 32 days). A catheter was placed in the central ear artery w2 cm from the base of the ear, under local anaesthetic (Xylocaine, 1% v/v lignocaine hydrochloride, Astra Pharmaceuticals, NSW, Australia) and conscious arterial pressure was measured for 30 min [20]. A 3 ml blood sample was taken, and centrifuged for collection of plasma, for measurement of PRA, by radioimmunoassay (ProSearch International Australia Pty. Ltd. VIC, Australia) and expressed as ng AngI/ml/h [21]. 2.3. Collection of placental tissue Dams were killed by an overdose of sodium pentobarbitone (Lethabarb, Pentobarbitone Sodium: 325 mg/ml, Virbac Pty Ltd, NSW, Australia) administered intravenously. A midline incision was made and the uterus exposed. The total number of fetuses and their positions were recorded. The fetuses were then removed and weighed. Placental tissue was detached from the fetus and membranes, the decidua was removed and placental tissue was weighed. Four placentas per mother were randomly collected at GA14, 21 and 28. These time points were selected as GA14 is two days after the chorion and allantois fuse thus is early in the vascular development of the chorioallantoic placenta [8], GA21 is the day after organogenesis is complete [8] and GA28 is when the placenta is maximally functional near term. Therefore these are likely to be useful time points for future experimental investigations. Placentas were sectioned mid-sagittally. Two placentas from each doe were immersion fixed in 10% buffered formalin for w48 h for

Fig. 3. Mid-sagittal cross-sectional labyrinthine area (A), mid-sagittal cross-sectional giant cell area (B) and mid-sagittal cross-sectional giant cell area-to-labyrinthine area ratio at gestational age (GA) 14, 21 and 28. Values expressed as estimated marginal mean  SEM. P < 0.05 for all dissimilar letters.

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Table 2 Structural correlates of placental function at gestation age (GA) 14, 21 and 28.

Volume density of trophoblast Volume of trophoblast (g) Volume density of fetal capillaries Volume of fetal capillaries (g) Volume density of maternal blood space Volume of maternal blood space (g) Surface density of trophoblast (cm2/g) Total surface area of trophoblast (cm2) Arithmetic mean barrier thickness trophoblast (mm)

GA14

GA21

GA28

0.694  0.031 a 0.209  0.070 a 0.215  0.023 a 0.066  0.064 a 0.091  0.014 a 0.026  0.051 a 369.048  51.510 a 108.797  177.632 a 20.360  1.796 a

0.679  0.036 a 0.882  0.081 b 0.274  0.027 ab 0.394  0.074 b 0.096  0.017 a 0.141  0.059 a 459.459  59.478 ab 597.440  205.111 a 16.011  2.199 ab

0.521  0.036 b 1.477  0.081 c 0.310  0.027 b 0.895  0.074 c 0.169  0.017 b 0.514  0.059 b 578.499  59.478 b 1760.389  205.111 b 11.185  2.074 b

Values expressed as estimated marginal mean  SEM. P < 0.05 for all dissimilar letters.

subsequent morphometric analysis, the other placentas were snap frozen in liquid N2 and stored at 70  C, for subsequent RNA extraction. 2.4. RNA extraction Total RNA was extracted using RNeasy extraction kits (Qiagen Pty. Ltd., Victoria, Australia) with additional DNAse treatment of the RNA (Qiagen Pty. Ltd., VIC, Australia) according to the manufacturer’s instructions. Gene expression for renin (Probe: 50 AAC CTC TAC GAC TCG GCG GAA TCC TC 30 , Primers FWD: 50 ACA CTG CCT GCG AGA TCC A 30 , REV: 50 CCG TCC CGT TCT CCA GGT A 30 ), AT1R (Probe: 50 TAT GGA ATA CCG CTG GCC CTT TGG C 30 , Primers FWD: 50 GCC ACT CTG GGC TGT CTA CAC 30 , REV: GCC CAA GCA ATC TTA CAC AGG TA 30 ), AT2R (Probe: 50 CAG GCC ATA CAC CAC ACA AGG GGA A 30 , Primers FWD: 50 AAA TCC CTG GCA AGC ATC TTA TA 30 , REV: 50 AAA AGT TGG CAA CGA GGA CAA 30 ) and 11b-HSD2 (Probe: 50 CTG GTC AAC AAC GCA GGT CAC AAC G 30 , Primers FWD: 50 CCA CCA GCA CGG GTC TGT 30 , REV: 50 AGC TCC ACA TCG GCC ACT AC 30 ), were determined using an Eppendorf MastercyclerÒ ep realplex real time PCR machine with accompanying analysis software (Eppendorf AG, Hamburg, Germany). PCR for all genes consisted of a primer/probe set and were multiplexed with primers for 18S, optimized for rabbit tissue. mRNA was quantified by the comparative cycle (DCT) method, using 18S as an internal control, as previously described [22]. 2.5. Placental morphometry Fixed placental tissue was processed and embedded in paraffin with the midsagittal surface face down. Adjacent mid-sagittal sections were cut at 5 mm and stained with Masson’s trichrome using standard protocols [23] or subjected to immunohistochemistry. 2.6. Immunohistochemistry Placental sections were double immunohistochemically labelled with monoclonal mouse anti human vimentin 3B4 (DakoCytomation, Denmark) to label fetal capillaries, and a monoclonal mouse anti bovine cytokeratin AE1/AE3 antibody (Millipore, NSW, Australia) to label trophoblast cells. A triple layer technique was used for labelling the primary antibody sequentially as previously described [24,25]. Mouse anti human vimentin 3B4 antibody was used at a dilution of 1 in 10 antibody to diluent (1% bovine serum albumin and 10% normal goat serum in phosphate buffered saline, pH 7.4) and a black precipitate was developed at the site of the antivimentin 3B4 staining by incubating with diaminobenzidine (DAB) (Sigma, St Louis, USA) with 2% ammonium nickel (II) sulphate (Sigma, St Louis, USA). Mouse anti bovine cytokeratin AE1/AE3 was used at a dilution of 1 in 25 antibody to diluent (1% bovine serum albumin and 10% normal goat serum in phosphate buffered saline, pH 7.4) and DAB without the nickel enhancement was used to develop a brown precipitate at the site of the AE1/AE3 anti-cytokeratin binding. A biotinylated goat anti-mouse secondary antibody IgG (DakoCytomation, Denmark) was used at a dilution of 1 in 200 in 1% BSA for both primary antibodies. Sections were counter stained with haematoxylin to label nuclei and red cytoplasmic stain to identify red blood cells. Two negative control sections were included in each immunohistochemical run. One section was used as a negative control for the anti-vimentin 3B4, and one section was used as a negative control for the AE1/AE3 anti-cytokeratin. Negative controls were subjected to the same protocol except the appropriate primary antibody for that control section was omitted and the section was incubated in the diluent (1% bovine serum albumin and 10% normal goat serum in phosphate buffered saline, pH 7.4) for the primary antibody incubation step. 2.7. Morphometric analyses Masson’s trichrome sections were analysed using a 4 objective lens and 2.5 ocular lens on an Olympus BH2 light microscope. Total mid-sagittal cross-sectional area and mid-sagittal cross-sectional area of placental giant cells were measured with a Video Image Analysis system using Video Pro 32 software (Leading Edge,

Australia) [24,25]. The mid-sagittal cross-sectional area of labyrinth was estimated by subtracting the mid-sagittal cross-sectional area of trophoblast giant cells from the total mid-sagittal cross-sectional area and then the proportion of mid-sagittal cross-sectional giant cell area-to-labyrinth area was calculated. Immunohistochemically double labelled sections were photographed with a 40 objective lens on a Zeiss Axioplan 2 imaging light microscope with accompanying AxioVision 2.05 Software (Zeiss Pty. Ltd., Germany). Images were then analysed using Image J 1.39u software (Image J 1.39u, National Institute of Health, USA) in order to estimate the volume density and volume of trophoblast, fetal capillaries and maternal blood space, surface density of trophoblast, total surface area of trophoblast and arithmetic mean barrier thickness of trophoblast as previously described [24,25]. Placental analyses were blinded and performed randomly in respect to gestational age. One section was selected at random and counted 5 times, in order to determine the coefficient of variation. The variation was <10%.

2.8. Statistical analysis Data are expressed as mean  SEM unless otherwise stated. Q–Q plot was used to determine if the data were normally distributed. One-way analysis of variance (ANOVA) with Bonferroni post-hoc corrections was used to test for statistical significance in maternal factors across gestation. Relationships between MAP, PRA, structural correlates of placental function, placental and fetal weights and placental gene expression across gestation were assessed by Pearson bivariate correlation analyses. Stepwise linear regression was used to determine factors associated with fetal weight, placental weight and structural correlates of placental function. Linear mixed model analyses were employed to assess the effect of gestational age on fetal and placental weights, structural correlates of placental function and placental gene expression with litter size as a covariate where appropriate. This analysis uses the mother as the subject and the fetal and placental parameters as repeated measures of the mother in order to account for the similarity in factors within mothers and differences between mothers across the gestational ages measured. This analysis also generates estimated marginal means which are used when representing these data. Sidak post-hoc corrections were used for pairwise multiple comparisons between treatment groups. Data were analysed using SPSS version 17 (SPSS, Chicago). P < 0.05 was considered statistically significant.

3. Results 3.1. Maternal measurements Maternal measurements are shown in Table 1. Maternal body weight increased from GA14 to GA21 (P < 0.05). There was no difference in mean arterial pressure (MAP) at GA0, GA14 or GA21 however MAP had decreased from GA21 to GA28 (P < 0.05) (Table 1). Maternal PRA at GA0, GA14 were similar, but by GA28 maternal PRA was significantly increased (Table 1; P < 0.05).

3.2. Fetal and placental weights Fetal weight (P < 0.01) and placental weight (P < 0.01) increased significantly with gestational age (Fig. 1). Fetal-to-placental weight ratio (P < 0.001) also increased across gestation due to the greater proportional increase in fetal weight (Fig. 1). Average litter size was not different at GA14 (5.4  1.1 pups/litter), GA21 (5.3  1.0 pups/ litter) or GA28 (6.6  1.2 pups/litter).

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3.3. Placental morphometry A representative micrograph of a Masson’s trichrome stained placental section used for measurement of mid-sagittal crosssectional labyrinthine area and giant cells is shown in Fig. 2. Midsagittal cross-sectional labyrinthine area was w824% (P < 0.01) greater at GA21 compared to GA14 but by GA28 it had decreased by w11% (P < 0.05) compared to GA21 (Fig. 3). Trophoblast giant cells were observed in greatest density at the decidual–placental interface. The mid-sagittal cross-sectional area of giant cells was w895% (P < 0.01) greater at GA21 compared to at GA14 but by GA28 had decreased to approximately the same area as at GA14 (P < 0.01; Fig. 3). There was no difference in mid-sagittal crosssectional giant cell area-to-labyrinthine area across gestation (Fig. 3). Estimates of volume densities (proportions) and volumes of structural correlates of placental function are shown in Table 2. Fetal capillaries occupied approximately one fifth of the labyrinth at GA14, increasing to nearly one third of this volume at GA28 (P < 0.05; Table 2). The volume density of trophoblast within the labyrinth decreased from w70% at GA14 to w50% at GA28 (P < 0.01). The surface density of trophoblast for exchange increased by nearly 57% (P < 0.05), while its total surface area increased w6 fold between GA14 and GA21 and w16 fold between GA14 and GA28 (P < 0.01; Table 2). Volume density of maternal blood space increased from w9% at GA21 to w17% (P < 0.01) at GA28, while its volume increased by 265% (P < 0.01) over the same period (Table 2). The arithmetic mean barrier thickness of trophoblast for exchange progressively decreased across gestation (P < 0.01; Table 2). 3.4. Placental gene expression Renin, AT1R, AT2R and 11b-HSD2 mRNA were all expressed in the placenta throughout gestation. All gene expression is presented relative to the calibrator group GA28. Placental renin mRNA became significantly less abundant with increasing gestational age with expression at GA28 w62% less than at GA14 (P < 0.05; Fig. 4). Placental AT1R mRNA became significantly greater with increasing gestational age with expression at GA28 w92% greater than at GA14 (P < 0.01; Fig. 4) Placental AT2R mRNA expression was w81% greater at GA21 compared to GA14 (P < 0.01; Fig. 4), but was not different between GA21 and GA28. Placental 11b-HSD2 mRNA was w74% greater at GA21 (P < 0.01) compared to GA14, but by GA28 had returned to levels similar to GA14 (Fig. 4). 3.5. Relationships between placental factors and fetal weight Relationships between structural correlates of placental function and gene expression with fetal and placental weights are shown in Table 3. Fetal and placental weights were positively correlated with each other and with PRA (GA0) and at time of tissue collection, placental expression of AT1R mRNA, volume of trophoblast, volume of fetal capillaries, volume density and volume of maternal blood space, surface density and surface area of trophoblast, and negatively correlated with placental renin mRNA and barrier thickness of trophoblast (Table 3). Mid-sagittal crosssectional labyrinthine area was negatively correlated with fetal weight and positively correlated with placental weight. Placental weight was also positively correlated with volume density of trophoblast. Fig. 4. Expression of placental renin mRNA (A), angiotensin type 1 receptor (AT1R) mRNA (B), angiotensin type 2 receptor (AT2R) mRNA (C) and 11b-hydroxysteroid dehydrogenase (11b-HSD2) mRNA at gestational age (GA) 14, 21 and 28. Expression

given relative to GA28; housekeeping gene was 18S. Values expressed as estimated marginal mean  SEM. P < 0.05 for all dissimilar letters.

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Table 3 Pearson bivariate correlations with fetal weight, placental weight and placental renin mRNA across gestation. Fetal weight R PRA (GA0) PRA (collection) Fetal weight Placental weight Placental renin mRNA Placental AT1R mRNA Placental AT2R mRNA Volume density trophoblast Volume trophoblast Volume fetal capillaries Volume density maternal blood space Volume maternal blood space Surface trophoblast Surface area trophoblast Barrier thickness trophoblast Labyrinthine area Ratio giant cell area-to-labyrinthine area

Placental weight P<

N

0.694 0.694

0.000 0.000

23 23

0.913 0.583 0.795

0.000 0.014 0.000 NS NS 0.000 0.000 0.001 0.000 0.013 0.000 0.009 NS 0.002

22 17 18

0.870 0.893 0.733 0.890 0.572 0.879 0.612 0.691

18 18 18 16 18 18 17 17

Placental renin mRNA was negatively correlated with PRA (GA0), fetal and placental weight, placental AT1R and AT2R mRNA and volume of trophoblast, and positively correlated with PRA at time of collection (Table 3). Stepwise linear regression was used to determine which were the most important factors associated with fetal weight, placental weight and structural correlates of placental function. Parameters entered into the models included MAP and PRA (GA0 and at time of collection), all structural correlates of placental function and placental gene expression. Nearly 94% of the variation in fetal weight could be explained by volume of maternal blood space (R2 ¼ 0.937; P < 0.01) and placental AT2R mRNA added to the model increased this by about 4% (R2 ¼ 0.988, P < 0.01). The variation in placental weight across gestation could largely be explained by volume of trophoblast (R2 ¼ 0.967; P < 0.01) plus barrier thickness of trophoblast (R2 ¼ 0.992; P < 0.01) and the ratio of giant cell area-to-labyrinthine area (R2 ¼ 0.998; P < 0.01). Linear regression models found that the volume of trophoblast was strongly associated with placental AT1R mRNA (R2 ¼ 0.888; P < 0.01) together with placental AT2R mRNA (R2 ¼ 0.953; P < 0.01). The volume of fetal capillaries and surface area of trophoblast for exchange were in part determined by placental AT1R mRNA (R2 ¼ 0.770 and R2 ¼ 0.520 respectively, both P < 0.01) and (R2 ¼ 0.436; P < 0.01), while the volume of maternal blood space was associated with placental AT1R mRNA (R2 ¼ 0.436, P < 0.01) plus placental 11b-HSD2 mRNA (R2 ¼ 0.844; P < 0.01). The surface density of trophoblast was largely determined by MAP (GA0) (R2 ¼ 0.936; P < 0.01) while barrier thickness of trophoblast was in part determined by MAP (GA0) (R2 ¼ 0.409, P < 0.01) plus placental 11b-HSD2 mRNA (R2 ¼ 0.409; P < 0.01). Mid-sagittal cross-sectional labyrinthine area and giant cell area were determined in part by placental 11b-HSD2 mRNA (R2 ¼ 0.640 and R2 ¼ 0.533 respectively; both P < 0.01). 4. Discussion The rabbit is a useful model for the study of fetal and placental development due to the similar hemodynamic changes that occur throughout gestation and placentation between the rabbit and human [5,20,26]. For the first time we have described the differentiation of the placenta and its expression of components of the RAS and 11b-HSD2 mRNA across gestation in the rabbit and demonstrated important relationships between maternal MAP and PRA and placental gene expression of

R

Placental renin mRNA P<

N

R

P<

N

0.621 0.621 0.913

0.002 0.002 0.000

22 22 22

0.689 0.689 .583 0.709

0.002 0.002 0.014 0.001

17 17 17 17

0.709 0.803 0.670 0.633 0.946 0.928 0.622 0.887 0.609 0.890 0.634 0.657 0.499

0.001 0.000 0.002 0.006 0.000 0.000 0.004 0.000 0.010 0.000 0.006 0.004 0.049

17 18 18 17 17 17 17 16 17 17 17 17 16

0.653 0.537

0.004 0.026 NS 0.039 NS NS NS NS NS NS NS NS

17 17

0.601

12

components of the RAS with structural correlates of placental function. As expected, fetal and placental weight and fetal weight-toplacental weight ratio increased with gestation. Fetal-to-placental weight ratio, a surrogate for placental efficiency, showed the most rapid increase in late gestation as from approximately mid gestation the fetus undergoes exponential growth while the placenta has achieved maximal functional capacity. Throughout gestation fetal weight was positively correlated with placental weight as seem in most mammalian species. Mid-sagittal crosssectional labyrinthine area (placental exchange region) increased from GA14 to GA21. As gestation progresses the placental labyrinth is increasingly occupied by fetal capillaries and trophoblasts invade the maternal vasculature to sequester a maternal blood supply, as indicated by an increase in the proportion and volume of maternal blood space, in order to achieve maximal placental functional capacity and hence fetal growth near term [9,10]. The fetal and placental growth curves described in this study are consistent with those described in the New Zealand White and Chinchilla rabbit breeds [27] and are similar to growth patterns described in other mammalian species including humans, pigs, sheep and monkeys, demonstrating that placental growth is most rapid during the first half of pregnancy, while during the second half of pregnancy the fetus undergoes substantial weight gain [28]. Representative micrographs (Fig. 5) of the rabbit placenta across gestation clearly show that placental differentiation predominates in the second half of gestation. This is demonstrated by increasing convolution of the exchange surface as reflected in the substantial increase in surface density, surface area and reduced trophoblast thickness. Together, these factors are important determinants of placental exchange capacity because they enable increases in the efficiency of placental diffusional transport processes [10]. Positive correlations between proportions and volume of trophoblast, fetal capillaries and maternal blood space and surface density and surface area of trophoblast with fetal and placental weight, as well as a negative correlation between diffusion distance and fetal and placental weight, have been described in both humans [29] and in guinea pigs [24], reflecting the importance of these structural correlates of placental function to fetal growth and development. Terminal trophoblast differentiation and increasing placental vascularisation by both fetal capillaries and maternal blood space occur during the second half of gestation which together enable the

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Fig. 5. Representative immunohistochemically double labelled sections demonstrating ontogenic change in differentiation of the rabbit placental labyrinth. These show increasing convolution of the exchange surface and vascularisation across gestation consistent with increasing functional capacity. Black ¼ anti-vimentin labelling of fetal capillaries, FC; Brown ¼ anti-cytokeratin labelling of trophoblast cells (T); red ¼ maternal and fetal red blood cells; MBS ¼ maternal blood space). All photographed with 40 objective lens at gestational age (GA) 14, (A), 21 (B) 28 (C) and negative control (D).

placenta to achieve large increases in nutrient and oxygen delivery efficiency without having to drastically increase its size [9,24,28]. A number of pregnancy complications and perturbations, including specific gene ablation, have been associated with poor placental vasculogenesis and impaired fetal growth [30,31]. The changes in rabbit placental structural architecture are comparable to the morphometric changes seen in humans where increased placental efficiency is achieved by a 10-fold increase in villous volume occupied by the vasculature, an increase in trophoblast surface area and a decrease in the maternal–fetal diffusion distance [10]. Our data demonstrate that the rabbit provides a good model for investigations into pregnancy complications associated with altered placental morphology. A distinct morphological difference between the rabbit and human placenta is the presence of giant cells in highest density at the feto-maternal interface from GA10 [32]. Total mid-sagittal cross-sectional area of trophoblast giant cells was found to be greatest at GA21, declining thereafter. These trophoblast giant cells are thought to contain glycogen and lipid [32] and have been shown to degenerate with increasing gestational age [6,32]. Initially, the origin of these placental giant cells was unclear but they are now considered to be trophoblastic cells from the fetal labyrinth that migrate within the maternal blood vessels and replace the endothelial cells [32]. Hence they appear to be important in the remodelling of spiral arteries [32,33] and may transport nutrients to the fetus [6,32,33]. In the rat, placental trophoblast giant cells are highly invasive [34], while in the mouse, giant cells produce hormones and cytokines [35], as well as invade and remodel the maternal vasculature [30]. The local placental RAS is thought to be important in placental angiogenesis and trophoblast invasion [36]. In the human, renin mRNA has been identified in the endometrium, choriodecidua and placental tissue [36] as well as around the spiral arteries, suggesting that renin may play a role in remodelling of blood vessels [37]. This is consistent with the decline in the expression of renin mRNA in late gestation after vascular remodelling and when the placenta is maximally functional. However, the relationship between mRNA expressions of components of the RAS to protein expression still remains to be determined. Maternal PRA undergoes a sustained increase from early in gestation in both the human [38] and rabbit [20]. The mechanisms involved in the interaction between maternal circulating renin and local placental RAS remain largely unknown but the increase in

maternal PRA coincides with the decline in placental renin mRNA expression suggesting a possible regulatory involvement between the two systems. Of considerable interest is the possibility that placental renin may enter the maternal circulation to affect maternal blood pressure. This was demonstrated in an experiment in mice in which a human renin transgene in the father, expressed by the placenta, resulted in human renin entering the maternal circulation, resulting in cleavage of human angiotensinogen, encoded by a transgene in the mother which induced maternal hypertension late in gestation [39]. Similarly, a study in New Zealand White rabbits showed that during late gestation, uteroplacental renin was involved in the maintenance of maternal blood pressure through its regulation of systemic vascular resistance, as well as suggesting a role for renin in the local regulation of uteroplacental blood flow [40]. The release of renin results in a cascade of events that leads to the production of angiotensin II. Angiotensin II acts via two receptors, angiotensin II receptor type 1 (AT1R) and angiotensin II receptor type 2 (AT2R). Actions of angiotensin II on the AT1R include increasing vascular tone and thereby blood pressure. The ontogenic expression of the angiotensin II receptors in the placenta is species dependent. In humans the AT1R is the major placental receptor and is present from early in gestation [41], while a greater proportion of AT2R are found in the rat [42] and rabbit [15]. The localisation and expression across gestation of placental AT1R suggests that this receptor is important in normal placental vascular development and function [43]. The physiological role of placental AT2R is less well understood but it may be involved in cellular growth and differentiation, including the inhibition of cell proliferation, apoptosis and potentially vasodilation [44]. The increased expression of the AT2R mRNA seen in the rabbit placenta late in gestation, coincides with the increases in volume densities of fetal capillaries and maternal blood spaces and attenuation of the trophoblast barrier, suggesting that it may be involved in some of these placental remodelling processes [14]. Indeed regression analyses in the current study support a role for both AT1R and AT2R in the differentiation of key components of the rabbit placenta in mid to late gestation when placental functional capacity is escalating as indicated by the exponential increase in fetal weight at this time. The correlations between structural correlates of placental function with maternal PRA and placental expression of renin across gestation suggest that the RAS may be important in placental

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invasion, differentiation and growth and that there may be an interaction between circulating renin derived from the mother with that from the placenta. A number of pregnancy complications, including those complicated by pre-existing hypertension and preeclampsia, show disruption to the maternal RAS [13], as well as the placental RAS [45], suggesting the role of the both the placental and maternal RAS in hypertensive pregnancies warrants further investigation. Tight control of fetal exposure to maternal glucocorticoids is vital for normal fetal development. During pregnancy maternal glucocorticoids circulate at high concentrations and would easily cross the placenta except for the presence of the enzyme 11b-HSD2. Placental 11b-HSD2 catalyses the reaction of active cortisol into its inactive metabolites, thus regulating fetal exposure to maternal glucocorticoids [17]. While these aid in the late maturation of various organ systems including the lung, gut and liver maturation [46], excess exposure, particularly during early and mid gestation, is detrimental to the fetus. Studies in both humans and animals have shown fetal exposure to maternal glucocorticoids adversely affects fetal development, in particular that of the kidney and brain [47]. 11b-HSD2 mRNA in the rabbit placenta was highly expressed across gestation particularly at GA21, just after completion of organogenesis, and then was lower late in gestation when the movement of cortisol across the placenta may be involved in organ maturation. In women, placental 11b-HSD2 is localized to the syncytiotrophoblast and has also been shown to have decreased activity in late gestation [48]. In conclusion, we have shown that structural correlates of placental function and placental gene expression in the rabbit placenta, along with the hemodynamic changes that occur in the rabbit mother are similar to those seen in the human, thus the rabbit provides a good model for perturbations of pregnancy that alter RAS activity or programme the fetus. This study has also identified suitable time points for the study of such perturbations across gestation. Furthermore, we have shown important relationships between structural correlates of placental function and components of the RAS. These relationships emphasise the vital role of this system in normal placental development and pregnancy outcome and highlight the need for further investigations into the role of the placental RAS in pregnancy complications that are associated with alterations in circulating maternal PRA, including chronic maternal hypertension and preeclampsia. The similarities in placental development and gene expression between the rabbit and human may pave the way for studies on the molecular mechanisms by which pre-existing maternal hypertension is a risk factor for preeclampsia. 5. Conflicts of interest The authors do not have any conflicts of interest to disclose. Acknowledgements This study was supported by the National Health and Medical Research Council of Australia Project Grant 490920, and a National Heart Foundation Grant-in-aid G03M1143. Assoc Prof KM Denton was supported by National Health and Medical Research Council Senior Research Fellowship 490918. We thank Gary Heinemann for performing the immunohistochemistry staining. References [1] Gude NM, Roberts CT, Kalionis B, King RG. Growth and function of the normal human placenta. Thromb Res 2004;114(5–6):397–407.

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