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Influence of Maternal Nutrition on Messenger RNA Expression of Placental Angiogenic Factors and Their Receptors at Midgestation in Adolescent Sheep¹

Rowett Research Institute,³ Bucksburn, Aberdeen AB21 9SB, United Kingdom Department of Animal and Range Sciences,⁴ North Dakota State University, Fargo, North Dakota 58105

	ABSTRACT

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Previous studies have shown that placental growth and pregnancy outcome are severely compromised in adolescent ewes overnourished to promote rapid maternal growth. Using this paradigm, the aim of the present study was to investigate expression of the major angiogenic factors and their receptors in the placenta at the onset of the most rapid phase of fetal growth. Singleton pregnancies to a single sire were established by embryo transfer, and thereafter, adolescent dams were offered a high or moderate nutrient intake predicted to induce compromised or normal fetoplacental size at term, respectively. Ovine-specific oligonucleotide probe and primer sets for several angiogenic factors and their receptors were developed for quantitative real-time reverse transcription-polymerase chain reaction determination of placentome mRNA expression at Day 81 of gestation. Total placentome weight and fetal weight were equivalent in high- compared with moderate-intake groups at this stage of gestation. Placentome expression of the angiogenic factors, vascular endothelial growth factor, angiopoietins 1 and 2, and nitric oxide synthase 3, were reduced in overfed ewes. Similarly, level of expression of vascular endothelial growth factor/vascular permeability factor receptor (FLT1) was less in overfed ewes. Thus, in the adolescent, maternal overnutrition has a negative impact on midgestation placental angiogenic factor/ receptor expression. This may impact placental vascularity and explain why uteroplacental mass, blood flow, and nutrient uptake are compromised in late pregnancy, resulting in low-birth-weight offspring.

growth factors, placenta

	INTRODUCTION

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The growth of the placenta precedes that of the fetus, and a strong correlation exists between placental mass and size at birth in all species studied [1–5]. The placenta is the organ through which respiratory gases, nutrients, and wastes are exchanged between the maternal and fetal systems. Thus, transplacental exchange provides for all the metabolic demands of fetal growth. Transplacental exchange is dependent on uterine and umbilical blood flow, and blood flow rates are, in turn, dependent largely on the vascularity of the placenta [5]. Therefore, factors that influence placental growth and vascular development have a dramatic impact on fetal growth and, thereby, on fetal and neonatal mortality and morbidity [5–7]. Evidence is beginning to emerge that inappropriate maternal nutrition is one of the major extrinsic and, potentially, modifiable factors influencing the development of the placental vascular bed [8].

Previous studies using our adolescent sheep paradigm, which replicates the adverse pregnancy outcome observed in growing human adolescents [9], have demonstrated consistently that overnourishing the singleton-bearing adolescent ewe promotes rapid maternal growth at the expense of the nutrient requirements of the gravid uterus. This results in a major reduction in placental mass and premature delivery of low-birth-weight lambs following spontaneous delivery near term [10–12]. In addition to the observed reduction in placental mass, these pregnancies are characterized during late gestation by attenuated absolute uteroplacental blood flows and nutrient uptakes (oxygen, glucose, and amino acids). This results in fetal hypoxia, hypoglycemia, hypoinsulinemia, and asymmetric growth restriction [12–14]. We hypothesize that these late-pregnancy events are preceded by alterations in placental angiogenesis/vascularity.

Thus, our goal in the present study was to focus on an earlier gestational time point, when the proliferative growth of the placenta is largely complete, but before the onset of rapid fetal growth—namely, Day 81 of gestation. Therefore, the aim was to investigate the gene expression of several major angiogenic factors (vascular endothelial growth factor [VEGF], angiopoietin 1 [ANGPT1] and angiopoietin 2 [ANGPT2], fibroblast growth factor 2 [FGF2], and nitric oxide synthase 3 [NOS3]) and angiogenic factor receptors, such as fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor [FLT1]), kinase insert domain receptor (a type III receptor tyrosine kinase [KDR, a VEGF receptor], endothelial tyrosine kinase (venous malformations, multiple cutaneous and mucosal [TEK, also known as TIE-2, an angiopoietin receptor]), and guanylate cyclase 1, soluble, ß3 (nitric oxide receptor [GUCY1B3]), in putatively compromised compared with normally growing placentae. For reviews concerning the function of these angiogenic factors in the placenta, see Redmer et al. [8] as well as Reynolds and Redmer [15].

	MATERIALS AND METHODS

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Animals and Experimental Design

Singleton pregnancies to a single sire were established by embryo transfer, and thereafter, adolescent dams were offered a high or moderate nutrient intake predicted to induce compromised or normal fetoplacental size at term, respectively. All procedures were as described previously [16] and were licensed under the U.K. Animals (Scientific Procedures) Act of 1986 and by the Rowett Research Institute's Ethical Review Committee.

Briefly, embryos from superovulated adult ewes (Border Leicester x Scottish Blackface), inseminated by a single sire, were recovered on Day 4 after estrus and transferred synchronously in singleton fashion into the uterus of recipient ewe lambs (Dorset Horn x Mule). Embryo transfer was carried out during October and November (midbreeding season), and the animals were housed in individual pens under natural lighting conditions at the Rowett Research Institute (57°N, 2°W). At the time of embryo transfer, the recipient ewe lambs were pubertal (age, 7 mo), with a mean live weight of 45 ± 0.6 kg and a body condition score [17] of 2.3 ± 0.02 U. Immediately following embryo transfer, recipient ewe lambs were evenly allocated to one of two nutritional treatments on the basis of live weight, body condition score, and ovulation rate at the time of transfer. Where possible, care also was taken to randomize for embryo source. Recipients were individually offered either a high (n = 18) or moderate (n = 18) level of a diet calculated to promote rapid or moderate maternal growth rates, respectively. The moderate dietary level was, in fact, a control group in that this level of dietary intake was predicted to optimize placental and fetal growth in this genotype. The complete diet supplied 10.2 MJ of metabolizable energy and 137 g/kg of crude protein and was offered in two equal feeds at 0800 and 1600 h daily. The diet contained 30% (w/w) coarsely milled hay, 50% barley, 10% molasses, 9% fishmeal, 0.3% salt, 0.5% dicalcium phosphate, and 0.2% of a vitamin-mineral supplement and had an average dry matter content of 86%. Animals offered moderate intakes received their entire ration immediately; those offered high intakes had the level of feed gradually increased over a 2-wk period until the level of daily feed refusal was approximately 15% of the total offered (equivalent to ad libitum intakes). The level of feed offered was reviewed three times weekly and adjusted, individually as and when appropriate, on the basis of body weight-change data (recorded weekly) and the level of feed refused (recorded daily). Body condition score was subjectively assessed on a five-point scale (1 = emaciated, 5 = obese;) [17] on six occasions during the present study. Transabdominal ultrasound at approximately Day 50 of gestation confirmed viable fetuses in 13 moderate- and 14 high-intake ewes.

Measurements at Autopsy

Pregnant ewes were killed at Day 81 of gestation. To determine placental cell proliferation (labeling index), a subset of ewes (n = 6 per group) was infused with bromodeoxyuridine (BrdU; 5 mg/kg body weight, dissolved in PBS to form a saturated solution of 16.7 mg/ml at pH 7.0) via a catheter inserted in the jugular vein exactly 1 h before lethal injection as previously described [18]. Animals were killed by i.v. administration of an overdose of sodium pentobarbitone (20 ml of Euthesate; 200 mg/ml of pentobarbitone; Willows Francis Veterinary, Crawley, U.K.), and exsanguination was carried out by severing the main blood vessels of the neck. The gravid uterus was removed immediately and opened. The fetus was killed by intracardiac administration of sodium pentobarbitone (3 ml of Euthesate). The umbilical cord was clamped, and the fetus was removed, dried, and weighed. The uteri were dissected to remove 8 or 12 large placentomes, which were weighed. Four or eight placentomes were then sliced into 7-mm cross sections and immersion-fixed either in Carnoys fixative for 6 h followed by 70% ethanol changed once after 24 h (BrdU-treated ewes only) or in 10% neutral buffered formalin for analyses not reported here. Four whole placentomes were snap-frozen in liquid nitrogen-cooled isopentane and subsequently stored at –80°C. All remaining placentomes were dissected and then weighed to calculate the total placentome weight. Fetal brain and liver were dissected and weighed, as was the maternal perirenal fat.

Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction

Messenger RNA levels for the range of angiogenic growth factors and their receptors (Table 1) were determined using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from an individual, representative whole placentome of each pregnancy using Tri-Reagent (Sigma-Aldrich Co. Ltd., Dorset, U.K.). The quality and quantity of total RNA were determined via capillary electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE). All reagents and procedures used for the real-time RT-PCR were purchased from and used as directed by Applied Biosystems (Warrington, Cheshire, U.K.) [19]. For each sample, approximately 30 ng of total RNA were reverse transcribed in triplicate using TaqMan Reverse Transcription Reagents and MultiScribe Reverse Transcriptase. TaqMan probes and primers sets were designed from species-specific sequences of genes using Primer Express Software (Applied Biosystems). The sequences of the FAM-labeled TaqMan probes and the forward and reverse primers are detailed in Table 1. Polymerization and amplification reactions for each RT were carried out in duplicate using 96-well PCR plates in a final volume of 10 µl with an ABI PRISM 7700 Sequence Detector (Applied Biosystems). Hybridization and polymerization were carried out at 60°C for, typically, 40 cycles. Quantification was determined using a relative standard curve method with different doses of a reference standard cDNA generated from RNA pooled from Day 81 placentomes. Individual placentome mRNA for each gene of interest was expressed relative to the sample's internal 18S RNA using 18S PDAR kit reagents from Applied Biosystems.

Data Analysis

Data were analyzed using the general linear models procedure of the Statistical Analysis System [20]. Differences between specific means were determined using the Bonferroni t-test [21]. Correlations between maternal weight gains or fetal weights and angiogenic factor expression were conducted by the CORR Procedure of SAS, and Pearson correlation coefficients are reported [20]. Data are preseneted as mean ± SEM throughout.

	RESULTS

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Dietary Induced Changes in Maternal Weightand Adiposity

The mean weekly maternal feed intakes from embryo transfer to Day 81 of gestation and the resulting changes in maternal weight and body condition score are shown in Figure 1. Maternal dietary intakes were elevated in high- compared with moderate-intake dams throughout the present study (P < 0.001). High dietary intakes promoted rapid changes in maternal live weight and adiposity, which reached statistical significance by Day 21 and Day 45 of gestation, respectively. Mean daily live weight gain was 67 ± 4 and 273 ± 14 g/day in the moderate- and the high-intake group, respectively (P < 0.001). At autopsy on Day 81 of gestation, the absolute (P < 0.001) and relative (P < 0.01) perirenal fat mass of the dams was markedly elevated in the high-intake group (Table 2).

View larger version (18K): [in this window] [in a new window]   FIG. 1. Feed intake (Top), weight change (Center), and body condition score (Bottom) of adolescent ewes from embryo transfer to slaughter on Day 81 of pregnancy. Ewes were individually offered either a high-level (n = 14) or a moderate-level (n = 13) diet calculated to promote a rapid or moderate maternal growth rate, respectively. Bars indicate the SEM

Dietary Effects on Placental and Fetal Growth, Placental Angiogenic Factor Expression, and Cell Proliferation

At Day 81 of gestation, maternal nutritional treatment group did not influence total and average placentome weight or placentome number (Table 2). Seven and nine fetuses were male in the moderate- and the high-intake group, respectively, and no difference was observed in the weight of the fetal body, brain, or liver at this stage of gestation (Table 2).

Placental mRNA expressions of VEGF, ANGPT1, ANGPT2, and NOS3 were significantly lower in the high- compared with the moderate-intake group at Day 81 of gestation (Fig. 2). Dietary intake did not alter placental mRNA expression of FGF2 (Fig. 2). Placental mRNA expression of FLT1 was decreased in the high-intake group, whereas expression was not affected by maternal dietary intake for KDR, TIE-2, and GUCY1B3 (Fig. 3). Irrespective of nutritional treatment group, the relative expressions of VEGF, ANGPT1, and ANGPT2 were significantly and negatively associated with maternal live weight gain (r = –0.53, –0.58, and –0.39, respectively; P < 0.05; n = 27). In addition, fetal weight was significantly and negatively correlated with VEGF expression (r = –0.39, P < 0.05). Furthermore, positive correlations were detected between the placental expression of a number of the angiogenic growth factors and their receptors (Table 3). The percentage of proliferating cells in the placentae of a subset of animals has been published previously [22]. Cell proliferation was substantially lower in the high- compared with the moderate-intake group (9.0% ± 0.9% vs. 12.6% ± 0.7% of nuclear area found to be BrdU positive, respectively; P < 0.01).

View larger version (17K): [in this window] [in a new window]   FIG. 2. Angiogenic factor mRNA expression in placentomes from adolescent ewes on Day 81 of pregnancy. Ewes were individually offered either a high- or moderate-level diet calculated to promote a rapid or moderate maternal growth rate, respectively. Bars indicate the SEM. **P < 0.01,* P < 0.05, NS, Not significant (P > 0.05)

View larger version (16K): [in this window] [in a new window]   FIG. 3. Angiogenic factor-receptor mRNA expression in placentomes from adolescent ewes on Day 81 of pregnancy. Ewes were individually offered either a high- or moderate-level diet calculated to promote a rapid or moderate maternal growth rate, respectively. Bars indicate the SEM. *P < 0.05. NS, Not significant (P > 0.05)

	DISCUSSION

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Previous studies have shown that placental growth and pregnancy outcome at term are severely compromised in adolescent ewes overnourished to promote rapid maternal growth [11, 12]. During late gestation, these pregnancies are characterized by major reductions in absolute uteroplacental blood flows, leading to attenuated fetal (umbilical) uptakes of oxygen, glucose, and amino acids as well as asymmetric growth restriction [12–14]. The data contained herein confirm our hypothesis that the aforementioned late-pregnancy events are preceded by alterations in placental angiogenic growth factor expression. Thus, at the midgestation time point that was studied, whole-placentome expressions of four of five angiogenic factors examined (VEGF, ANGPT1, ANGPT2, and NOS3) and of one of four of their receptors (FLT1) were significantly attenuated in the overnourished compared with the moderate-intake control group. These alterations in placental angiogenic factor expression preceded any change in the total wet mass of the placentomes, which was equivalent in the high- and the moderate-intake groups. Nevertheless, this attenuated angiogenic factor expression (together with the previously reported reduction in placental proliferation [22]) suggests that these placentae were already on a different hemodynamic and growth trajectory. We were unable to quantify histologically the vascularity at this midgestation time point because of how the samples were initially processed. Recently, however, we have measured uterine blood flow in vivo in adolescent ewes exposed to identical nutritional regimes. At Day 90 of gestation, blood flow in the uterine artery supplying the gravid uterine horn was 170 ± 38 versus 386 ± 37 ml/min in the high- (n = 4) compared with the moderate-intake (n = 5) group, respectively (unpublished observations). Taken together, these observations of reduced angiogenic growth factor expression and maternal placental blood flow imply that impaired development of the placental vascular bed is an early defect in these nutritionally compromised adolescent pregnancies.

Significant negative correlations between maternal weight gain and placental mRNA expression for several of the key angiogenic factors detailed above indirectly suggest that the level of nutrient intake may determine the placental angiogenic factor expression and, therefore, the "vascular readiness" of the placenta needed to support the explosive growth of the fetus that occurs during the last third of gestation. In the present study, fetal weights were equivalent between treatments at Day 81, yet we have repeatedly shown that both placental and fetal weight is significantly reduced at term in the overnourished ewes [10, 12]. This suggests that the placenta in overnourished dams likely does not have either the size or the vascular exchange capacity for adequate nourishment of the fetus during its final growth phase. Unfortunately, the mechanism by which nutrient intake level affects angiogenic factor expression cannot be discerned from the present results. One possibility worth further examination is that nutritionally induced suppression of the major sex steroids in the overnourished dams is influencing both uteroplacental angiogenesis and blood flow. We previously reported that progesterone is significantly attenuated throughout gestation in the overnourished dams [23, 24], and estrogen concentrations likely are similarly affected. In addition, we have shown previously that estradiol upregulates expression of angiogenic factors in uterine endometrium [25, 26], suggesting that nutritional factors affecting circulating steroid levels could, in turn, influence angiogenic factor production. Alternatively, because angiogenic factors typically are expressed in response to a physiological need for angiogenesis (e.g., wound healing, hypoxia), maternal supply and availability of circulating nutrients may "program" vascular development in the placenta. Therefore, placental vasculature in overnourished ewes may develop to a lesser extent both because of an excess of available nutrients and because the fetoplacental unit fails to "detect" a need for a normal state of vascular readiness. This idea is obviously supported by the reduced placental angiogenic factor production in the present study as well as by the reduced uterine arterial blood flow in this same model, as mentioned above (unpublished observations), in overnourished ewes at approximately Day 90 of gestation.

We also have observed nutritional effects on placental angiogenic factor expression in adult ewes (unpublished observations). Expression levels for VEGF and FLT1 were not affected, whereas the VEGF-receptor KDR was modestly but significantly reduced in placentomes collected on Day 90 from ewes receiving 60% of a maintenance diet (VEGF: 1942 ± 386 vs. 2163 ± 63 for maintenance and reduced dietary intake, respectively; FLT1: 2332 ± 278 vs. 2372 ± 215, respectively; KDR: 2483 ± 247 vs. 2037 ± 188, respectively). Similar to the present results, differences in fetal weights at this time point were not detected, but by Day 130 of gestation, fetal weight was reduced.

To our knowledge, comparable data for nutritionally perturbed pregnancies in the human are not currently available because of the obvious difficulties in obtaining anything other than late-gestation or term tissues. However, attenuated placental VEGF expression has been reported in both preeclamptic and severely intrauterine growth-restricted pregnancies [27, 28], implying a central role for this angiogenic factor in the etiology of two types of compromised placentae.

Interestingly, Regnault et al. [29] reported contrasting placental angiogenic factor expression in compromised ovine pregnancies induced by acute versus chronic hyperthermia. Acute exposure of ewes to hyperthermia from Day 40 to Day 55 of gestation resulted in elevated expressions of VEGF, ANGPT1, ANGPT2, and TIE-2 in the fetal placenta that was suggested to reflect a compensatory mechanism aimed at maintaining normal placental development by enhancing angiogenesis. In contrast, chronic exposure to hyperthermia for approximately 80 days resulted in decreased expressions of VEGF and FLT1 in the fetal placenta, and of TIE-2 in both the fetal and maternal placenta, at Day 135 of gestation. This chronic response is similar to our observations in chronically overfed adolescents, and it suggests that prolonged physiological challenges during pregnancy can affect angiogenic factor production and, seemingly, vascular growth.

A unique observation in the present study is that expressions of VEGF, ANGPT1, ANGPT2, and NOS3 are significantly correlated with the message expressions of their major receptors. These data emphasize that receptors for the major angiogenic factors also play key roles in regulating vascular growth in the placenta.

The FGF2 gene expression was not significantly affected by overnutrition in the present study. The FGFs are unique among the major angiogenic growth factors in that they are pleiotropic and influence not only angiogenesis but also various other developmental and differentiated functions [30]. For example, FGFs have been shown to stimulate nitric oxide production (a major local vasodilator), which in turn has been shown to mediate estrogen-induced increases in uterine and placental blood flow and/or vascularization [25, 26, 31–34]. It is not clear why, in the present study, FGF2 expression was not affected by overnutrition but other key angiogenic factors were. Because FGF2 binds to extracellular matrix components, regulation of FGF2 expression might become secondary to mechanisms that release FGF2 in remodeling of the extracellular matrix during tissue growth.

In summary, the present study provides new information regarding expression of important angiogenic factors and their receptors in the overnourished adolescent ewe paradigm at a critical time point, when the fetus is approaching the onset of rapid fetal growth. These data show that by Day 81 of gestation, overnutrition inhibited a range of angiogenic factors known to regulate placental vascular growth. Thus, the data support our hypothesis that impaired angiogenesis and development of the vascular bed is an early defect in these nutritionally compromised pregnancies.

	ACKNOWLEDGMENTS

 
The authors wish to acknowledge Dr. Richard Lea and Lisa Hannah for assistance with the BrdU immunohistochemistry, Drs. Russell Anthony and Timothy Regnault for the ovine ANGPT1 and ANGPT2 nucleic acid sequences, and Dr. Mary Lynn Johnson for help with cloning and sequencing of other ovine angiogenic factor/receptor genes.

	FOOTNOTES

 
¹ Supported by the Scottish Executive Environment and Rural Affairs Department as part of the core funding to the Rowett Research Institute (J.M.W.) and supported in part by NIH grant HL64141 and Hatch Project ND01705 (D.A.R. and L.P.R.).

² Correspondence: Dale A. Redmer, Dept. of Animal and Range Sciences, 187 Hultz Hall, North Dakota State University, Fargo, ND 58105-5727. FAX: 701 231 7590; dale.redmer{at}ndsu.edu

Received: 22 October 2004.

First decision: 15 November 2004.

Accepted: 18 November 2004.

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				K. A Vonnahme, D. A Redmer, E. Borowczyk, J. J Bilski, J. S Luther, M. L. Johnson, L. P Reynolds, and A. T Grazul-Bilska Vascular composition, apoptosis, and expression of angiogenic factors in the corpus luteum during prostaglandin F2{alpha}-induced regression in sheep. Reproduction, June 1, 2006; 131(6): 1115 - 1126. [Abstract] [Full Text] [PDF]

				L. P. Reynolds, J. S. Caton, D. A. Redmer, A. T. Grazul-Bilska, K. A. Vonnahme, P. P. Borowicz, J. S. Luther, J. M. Wallace, G. Wu, and T. E. Spencer Evidence for altered placental blood flow and vascularity in compromised pregnancies J. Physiol., April 1, 2006; 572(1): 51 - 58. [Abstract] [Full Text] [PDF]

				L. P. Reynolds, P. P. Borowicz, K. A. Vonnahme, M. L. Johnson, A. T. Grazul-Bilska, D. A. Redmer, and J. S. Caton Placental angiogenesis in sheep models of compromised pregnancy J. Physiol., May 15, 2005; 565(1): 43 - 58. [Abstract] [Full Text] [PDF]

				J. M. Wallace, T. R. H. Regnault, S. W. Limesand, W. W. Hay Jr, and R. V. Anthony Investigating the causes of low birth weight in contrasting ovine paradigms J. Physiol., May 15, 2005; 565(1): 19 - 26. [Abstract] [Full Text] [PDF]

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