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BOR - Papers in Press, published online ahead of print October 18, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.054684
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BIOLOGY OF REPRODUCTION 76, 259–267 (2007)
DOI: 10.1095/biolreprod.106.054684
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Placental Growth Throughout the Last Two Thirds of Pregnancy in Sheep: Vascular Development and Angiogenic Factor Expression1

Pawel P. Borowicz , Daniel R. Arnold , Mary Lynn Johnson , Anna T. Grazul-Bilska , Dale A. Redmer , and Lawrence P. Reynolds 2

Center for Nutrition and Pregnancy, and Department of Animal and Range Sciences, North Dakota State University, Fargo, North Dakota 58105-5727

ABSTRACT

Morphometric methodologies were developed and applied to investigate the patterns of vascular development in maternal (caruncular; CAR) and fetal (cotyledonary; COT) sheep placentas throughout the last two thirds of gestation. We also examined the expression levels of the major angiogenic factors and their receptors in CAR and COT sheep placentas. Although the vascularity of the CAR tissues increased continuously from Day 50 through Day 140 of pregnancy, those of the COT tissues increased at about twice the instantaneous rate (i.e., the proportionate increase/day) of the CAR. For CAR, vascularity increased 2-fold from Day 50 through Day 140 via relatively small increases in capillary number and 2- to 3-fold increases in capillary diameter. For COT, the increased vascularity resulted from a 12-fold increase in capillary number associated with a concomitant 2-fold decrease in capillary diameter. This large increase in fetal placental capillary number, which was due to increased branching, resulted in 6-fold increases in total capillary cross-sectional area and total capillary surface, per unit of COT tissue. Different patterns of expression of the mRNAs for angiogenic factors and their receptors were observed for CAR and COT. The dilation-like angiogenesis of CAR was correlated with the expression of vascular endothelial growth factor receptor-1 (FLT1), angiopoietin-2 (ANGPT2), and soluble guanylate cyclase (GUCY1B3) mRNAs. The branching-like angiogenesis of COT was correlated with the expression of vascular endothelial growth factor (VEGF), FLT1, angiopoietin-1 (ANGPT1), ANGPT2, and FGF2 mRNAs. Monitoring the expression of angiogenic factors and correlating the levels with quantitative measures of vascularity enable one to model angiogenesis in a spatiotemporal fashion.

growth factor, placenta, uterus

INTRODUCTION

All of the respiratory gases, nutrients, and waste that are exchanged between the maternal and fetal systems are transported via the placenta [1–3]. Thus, the importance of transplacental exchange in supplying the metabolic substrates required for fetal growth has long been recognized [1–3]. The importance of placental function is probably best exemplified by the close relationship between fetal weight, final placental size, and uterine and umbilical blood flows in many mammalian species [3, 4].

The establishment of functional fetal and placental circulation systems is one of the earliest events in embryonic/placental development [2]. It has been suggested that the large increase in transplacental exchange, which supports the exponential rate of fetal growth during the last half of gestation, depends primarily on the dramatic growth of the placental vascular beds and the resultant large increases in uterine and umbilical blood flows [3]. Thus, factors that influence placental vascular development will have a dramatic impact on fetal growth and development, and thereby will affect neonatal survival and growth [3, 5, 6].

Vascular growth of maternal (caruncular; CAR [specifically, the caruncular portion of the endometrium]) and fetal (cotyledonary; COT [specifically, the cotyledonary portion of the chorioallantois]) placental tissues begins early in pregnancy and continues throughout gestation [3]. Stegeman [7] have reported that, in sheep, the vascular density of maternal placental tissues increases substantially from Day 40 through midgestation, and more slowly thereafter. However, the vascular density of the fetal cotyledons remains relatively constant until midgestation in sheep, and increases dramatically thereafter [7–9]. These data are consistent with the dramatic increases that have been reported for uterine and umbilical blood flows, and also with data indicating that umbilical blood flow increases more rapidly, on a proportional basis, than uterine blood flow during the last half of gestation [3, 10].

Angiogenesis is the formation of the new vascular beds, and it is a critical process for the growth and development of all tissues, including the placenta [11, 12]. The search for potential regulators of angiogenesis has led to the identification of the major angiogenic factors, and their role in angiogenesis has been established in numerous in vitro and in vivo studies, including those using gene knockouts in mice [13–16]. These major angiogenic growth factors include the vascular endothelial growth factor (VEGF) family and its major transmembrane tyrosine kinase receptors (FLT1 and KDR), basic fibroblast growth factor (FGF2) [17], angiopoietins and their tyrosine kinase receptor (TEK), endothelial nitric oxide synthase (NOS3), the NO receptor, soluble guanylate cyclase (GUCY1B3), and hypoxia inducible factor-1 (HIF1A).

The purpose of the present study was to evaluate the pattern of placental vascular development during the last two thirds of pregnancy in sheep, and to relate it to the patterns of mRNA expression of the major angiogenic factors and their receptors. To accomplish this, we developed histomorphometric methodologies and measured the placental expression of angiogenic factors using real-time, quantitative PCR.

MATERIALS AND METHODS

Animal Treatment and Tissue Collection

All procedures involving animals were approved by the North Dakota State University Institutional Animal Care and Use Committee. Crossbred ewes (primarily Rambouillet and Targhee crossbreds) were observed for signs of behavioral estrus twice daily using vasectomized rams, and were placed with an intact ram during estrus. Only those ewes that were observed to breed with the ram were included in the study, and the day of breeding was designated as Day 0 of pregnancy. Ewes were fed a ration of mixed forage and cracked corn, which was designed to meet the nutritional requirements of pregnant ewes [18], and had free access to a salt-mineral mixture and to water.

Gravid uteri were obtained from ewes on Day 50, 70, 90, 110, 130, or 140 after mating (gestation length in sheep is approximately 145 days; n = 5 per day, except for Day 90, for which n = 2). All the ewes were carrying either singles or twins, and the number of fetuses did not vary (P > 0.1) among the groups with an average of 1.53 ± 0.09 (±SEM) fetuses per ewe. Three representative placentomes from the gravid uterine horn, close to the fetus, were obtained from each ewe and separated into maternal CAR and fetal COT portions as described previously [19]. These CAR and COT tissues were snap-frozen in liquid nitrogen and stored at –80°C until used for RNA extraction. Additional placentomes from the same region of the uterus (i.e., the gravid uterine horn, near the fetus) were fixed by perfusion with Carnoy solution, as described below.

Measurement of Placental Vascularity (Angiogenesis)

To provide a quantitative description of vascular growth, we developed the following perfusion fixation procedure: for each ewe, several of the placentomes near the fetus were fixed with Carnoy solution by perfusion of the main arterial vessel(s) supplying the CAR or COT (i.e., by perfusion of a branch of the uterine or umbilical artery, respectively). These perfusion procedures were similar to those we have described previously [20–23]. After fixation, the placentomes were perfused with a vascular casting resin (Mercox; Ladd Industries, Williston, VT), embedded in paraffin, sectioned at 6-µm thickness, and stained with hematoxylin and periodic acid-Shiff (H&PAS) reagent using procedures reported previously [12, 24]. Photomicrographs were taken at 200x magnification using a Nikon DXM 1200 digital camera (Fryer, Chicago, IL). An additional set of pictures was taken using phase-contrast, to evaluate the quality of perfusion; a representative bright-field image and its phase-contrast image are presented in Figure 1. In addition, representative micrographs of stained histological sections from tissues perfused via the CAR or COT arteries are presented in Figure 2.


Figure 76021301
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  		FIG. 1. Brightfield (A) and phase contrast (B) photomicrographs of the same histological section of Mercox-perfused sheep COT at Day 140 of gestation. Arrows indicate how the luminescent plastic, which fills the capillaries (B), corresponds with the capillary vessels seen under brightfield in A. H&PAS, original magnification x400.


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  		FIG. 2. Photomicrographs of Mercox-perfused CAR (left-hand side) and Mercox-perfused COT (right-hand side) histological sections. Day 140 of gestation. Note how the fixed-perfused capillaries remain open on CAR (arrowheads) and COT (arrows). H&PAS, original magnification x400.

Vascularity was determined by image analysis (Image-Pro Plus, version 5.0; Media Cybernetics, Houston, TX). For each ewe, 20 areas (64 326 µm2 each) per placentome were analyzed, with the following parameters determined for each section and for each placental tissue type (i.e., for both the CAR and COT tissues): tissue area, shrinkage area (effect of fixation subtracted from the tissue area), cross-sectional capillary area density (CAD, total capillary area as a proportion of tissue area), capillary number density (CND, total number of capillaries per unit of tissue area), and capillary surface density (CSD, total capillary circumference per unit of tissue area). Although this latter parameter, CSD, actually represents the circumference of the capillary cross-sections, it is nevertheless proportional to their surface area [19]. To provide a measure of average capillary size, we also calculated the average cross-sectional area per capillary (APC) for CAR and COT by dividing the CAD by the CND.

Quantification of Placental Expression of mRNA for the Major Angiogenic Factors

The mRNA levels for ten angiogenic factors and their receptors (VEGF, FLT1, KDR, FGF2, ANGPT1, ANGPT2, TEK, NOS3, GUCY1B3, and HIF1A), as well as for ovine 18s mRNA were determined using quantitative real-time RT-PCR, as validated in our laboratories and described in a recent report [25]. Analyses were conducted using TaqMan reagents and procedures purchased from and recommended by Applied Biosystems (Foster City, CA).

Total cellular RNA (tcRNA) from frozen CAR and COT tissues was extracted using TriReagent (Molecular Research Center, Cincinnati, OH). The quality and quantity of the tcRNA was determined by capillary electrophoresis by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE). For each sample, approximately 30 ng of tcRNA was reverse-transcribed using TaqMan reverse transcription reagents and MultiScribe reverse transcriptase, as recommended by Applied Biosystems. TaqMan Universal PCR Master Mix was used in combination with TaqMan probes and primer sets that were designed from species-specific sequences of genes using the Primer Express Software (Applied Biosystems), as described previously [25].

In brief, polymerization/amplification reactions were performed in 96-well PCR plates sealed with optically clear adhesive covers using the Applied Biosystems ABI PRISM 7000 sequence detector. Hybridization and polymerization were performed at 60°C for approximately 40 cycles. For each sample, the fluorescence amplification plot showed the log change in fluorescence vs. cycle number, which allowed the determination of the threshold cycle (CT) in the exponential phase of amplification, in which none of the reaction components was limiting [25]. A standard curve of the resultant CT values was generated from different doses of a reference sample (from pooled late-pregnancy sheep placentomal tissues) vs. the log of the dose, and the relative quantities of the unknowns were determined by comparing their CT values to the standard curve. The ratios of all the mRNA values to their own 18s RNA values were used for the quantification of gene expression.

Statistical Analysis

For measurements of CAR and COT vascularity as well as for the expression of angiogenic factors, the data were analyzed using the PROC GLM program of SAS [26]. Initially, the model included day of gestation, tissue (CAR vs. COT), and the day of gestation by tissue interaction, with tissue nested within the animal as the error term for evaluating tissue differences. However, as the patterns of vascular growth differed dramatically between CAR and COT tissues, as indicated by a highly significant day-by-tissue interaction (P < 0.001 to P < 0.02 for all vascularity variables) and by the regression analysis (Fig. 3), we decided to analyze CAR and COT separately over days of gestation.


Figure 76021303
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  		FIG. 3. Regressions of vascularity measures for CAR and COT throughout the last two thirds of gestation in sheep. CAD, capillary area density (%) (total capillary area as a proportion of tissue area); CND, capillary number density (total number of capillaries per tissue area); CSD, capillary surface density in µm (total capillary circumference per tissue area); APC, area per capillary in µm2 (average cross-sectional area per capillary).

The data were then modeled mathematically using linear and non-linear regression procedures. For each variable, including vascularity parameters and mRNA levels, we evaluated linear, quadratic, cubic, exponential, and sigmoidal regressions against day of gestation; the regression chosen as having the best fit to the data was the one with the greatest R2 value. For those cases in which two models had the same R2, the simpler model was chosen (i.e., linear rather than quadratic, quadratic rather than cubic, etc.). In addition, we performed a simple correlation analysis of the measures of CAR and COT angiogenesis (CAD, CND, CSD, and APC) with the CAR and COT mRNA levels for angiogenic factors using the PROC CORR program of SAS.

We and others have previously used all of these statistical methods to model changes in fetal and placental growth, uterine and umbilical blood flows, nutrient fluxes across the placenta, transplacental clearance of water, placental expression of angiogenic factors, and oxygen consumption by the gravid uterus [3, 10, 19, 27, 28].

RESULTS

The perfusion-fixation methodology we developed gave very good perfusion of the placental capillary beds, resulting in readily visible capillaries (Figs. 1 and 2). Two criteria indicate that the size of these capillaries was not affected by the perfusion: 1) in all of the histological sections, the capillaries appeared to be intact (Fig. 2); and 2) the size of the capillaries, as reflected by their average diameter, was well within the range reported for a variety of tissues (Table 1).


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  		TABLE 1. Average CAR and COT capillary diameter (µm) throughout gestation in sheep.

Using this perfusion technique, we evaluated by image analysis the patterns of vascular growth during the last two thirds of pregnancy in sheep placentas. For the CAR portion of the placenta, CAD and CND increased exponentially (CAD = 5.336 e0.01154t; CND = 4.484 e0.00372t, where t = time in days), whereas CSD increased linearly (CSD = 608.154 + 4.958t; P < 0.02 to P < 0.001; Fig. 3). In contrast, for the COT portion of the placenta, CAD, CND, and CSD all increased exponentially (CAD = 0.838 e0.02093t; CND = 0.270 e0.02916t; CSD = 156.015 e0.02004t; P < 0.001 to P < 0.0001; Fig. 3).

In addition, although the CAD for CAR and COT increased exponentially (R2 = 0.59 and 0.81, respectively; P < 0.01) from Day 50 until Day 140 of pregnancy, the proportional rate of increase was greater for COT (5.4-fold, 2.09% per day; Fig. 3) than CAR (3.1-fold, 1.15% per day; Fig. 3). CAR CND also increased exponentially (R2 = 0.15; P < 0.02) but only slightly (0.4% per day; Fig. 3) throughout gestation, whereas for COT, the CND increased exponentially (R2 = 0.90; P < 0.01) and dramatically (2.9% per day; Fig. 3). In addition, the CSD of CAR capillaries increased linearly (R2 = 0.15; P < 0.02; Fig. 3) by 1.8-fold from Day 50 to Day 140 of gestation, whereas the CSD of COT increased exponentially (R2 = 0.91; P < 0.01) and dramatically (5.1-fold; Fig. 3). Lastly, from Day 50 to Day 140 of gestation, APC increased (P < 0.01) by 2.2-fold for CAR but decreased (P < 0.01) by 2.2-fold for COT (Fig. 3).

Using these regression models, we developed an empirical model of placental angiogenesis that explains the observed changes in vascularity (Fig. 4). The empirical model suggests that CAR vascularity increases primarily via an increase in capillary diameter. In contrast, COT vascularity reflects primarily increases in capillary number and complexity. The increased number and complexity of the fetal compared with the maternal microvasculature was reflected by a 2-fold greater surface area per unit of tissue area (i.e., CSD) for the COT compared with the CAR capillaries by Day 140 of gestation (Fig. 3).


Figure 76021304
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  		FIG. 4. Models of vascular growth and remodeling in CAR and COT tissues from Day 50 to Day 140 of pregnancy in sheep. Single upward arrows indicate moderate increases; double upward arrows indicate large increases.

For angiogenic factors, different patterns of mRNA expression were observed for the maternal (CAR) compared with the fetal (COT) placental tissues. The most dramatic changes in vascular growth and expression of mRNA for angiogenic factors occurred in COT. For example, although VEGF mRNA increased quadratically for both CAR (R2 = 0.71, P < 0.001) and COT (R2 = 0.85, P < 0.004), CAR VEGF mRNA expression peaked about Day 130, whereas that of COT was elevated between Day 90 and Day 130 of gestation (Fig. 5). FLT1 mRNA expression increased linearly for CAR (R2 = 0.49, P < 0.0001) and COT (R2 = 0.49, P < 0.01), although the rate of increase was greater for CAR (1.3%/day for CAR vs. 0.5%/day for COT). The increase in KDR mRNA expression for CAR followed a sigmoid pattern (R2 = 0.27, P < 0.008), whereas there was no significant change in KDR mRNA expression for COT (Fig. 5).


Figure 76021305
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  		FIG. 5. Expression of VEGF (A), KDR (B), and FLT1 (C) mRNAs in the sheep maternal CAR and fetal COT placentas throughout the last two thirds of gestation. In A, within a tissue, means with different superscripted letters are significantly different at P < 0.06 for CAR and P < 0.09 for COT. On Days 110, 130 and 140 CAR was different from COT (P < 0.03). In B, within a tissue, means with different superscripted letters are significantly different at P = 0.2 for CAR or are significantly different at P < 0.03 for COT. On Day 110 and Day 130, CAR was different from COT (P < 0.06). In C, within a tissue, means with different superscripted letters are significantly different at P < 0.08 for CAR and P < 0.06 for COT. On Days 50, 70, 90, 110, and 140, CAR was different from COT (P < 0.07).

Basic FGF mRNA expression did not change significantly in CAR but increased exponentially in COT (R2 = 0.32, P = 0.0025; Fig. 6). In contrast, the levels of NOS3 and GUCY1B3 mRNA expression did not change significantly in CAR and COT placental tissues throughout the last two thirds of gestation (Fig. 7).


Figure 76021306
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  		FIG. 6. Expression of FGF2 mRNA in the sheep maternal CAR and fetal COT placentas throughout the last two thirds of gestation. Within a tissue, means with different superscripted letters are significantly different at P = 0.3 for CAR or are significantly different at P < 0.01 for COT. On Days 50, 70, 110, and 130, CAR was different from COT (P < 0.04).


Figure 76021307
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  		FIG. 7. Expression of NOS3 (A) and GUCY1B3 (B) mRNAs in the sheep maternal CAR and fetal COT placentas throughout the last two thirds of gestation. In A, within a tissue, means with different superscripted letters are significantly different at P < 0.04 for CAR. On Day 140, CAR was different from COT (P < 0.008). In B, within a tissue, means with different superscripted letters are significantly different at P < 0.07 for CAR. CAR was not different from COT (P = 0.11).

ANGPT1 mRNA expression changed significantly in both CAR and COT (P < 0.005, R2 = 0.3 and P < 0.001, R2 = 0.47, respectively), and the relative rate of increase was similar for CAR and COT (around 1%/day). ANGPT2 mRNA expression changed significantly in both CAR and COT (P < 0.0001, R2 = 0.59 and P < 0.008, R2 = 0.3, respectively) and the relative rate of increase was greater for CAR than for COT (2.2%/day vs. 1.6%/day respectively). The levels of TEK mRNA expression remained constant in both CAR and COT throughout the last two thirds of gestation (Fig. 8).


Figure 76021308
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  		FIG. 8. Expression of the ANGPT1 (A), ANGPT2 (B), and TEK (C) mRNAs in the sheep maternal CAR and fetal COT placentas throughout the last two thirds of gestation. In A, within a tissue, means with different superscripted letters are significantly different at P < 0.09 for CAR and P < 0.05 for COT. On Days 70, 90, 110, and 140, CAR was different from COT (P < 0.02). In B, means with different superscripted letters are significantly different at P < 0.07 for CAR and P < 0.06 for COT. On Days 110 and 130, CAR was different from COT (P < 0.012). In C, within a tissue, means with different superscripted letters are significantly different at P = 0.1 for CAR and P = 0.25 for COT. CAR was not different from COT (P = 0.22).

Lastly, HIF1A mRNA expression decreased quadratically for both CAR and COT (P < 0.003, R2 = 0.32 and P < 0.06, R2 = 0.17, respectively); HIF1A mRNA peaked in CAR around Day 70 and decreased thereafter, while that in COT decreased from Day 50 through Day 140 (Fig. 9).


Figure 76021309
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  		FIG. 9. Expression of HIF1A mRNA in the sheep maternal CAR and fetal COT placentas throughout the last two thirds of gestation. Within a tissue, means with different superscripted letters are significantly different at P < 0.09 for CAR and P < 0.02 for COT. On Days 70 and 110, CAR was different from COT (P < 0.01).

Tables 2 and 3 show the correlations between the vascularity measurements and angiogenic factor mRNA expression levels for CAR and COT. For CAR, CAD was correlated with FLT1 (R = 0.54, P < 0.001), ANGPT2 (R = 0.50, P < 0.003), and GUCY1B3 (R = 0.34, P < 0.05) mRNA expression, CND was negatively correlated with HIF1A mRNA expression (R = –0.41, P < 0.02), and APC was correlated with FLT1 (R = 0.46, P < 0.007), KDR (R = 0.37, P < 0.03), and ANGPT2 (R = 0.57, P < 0.0006) mRNA expression. For COT, CAD was correlated with VEGF (R = 0.46, P < 0.008), FLT1 (R = 0.36, P < 0.03), ANGPT2 (R = 0.54, P < 0.001), FGF2 (R = 0.37, P < 0.03) and HIF1A (R = –0.38, P < 0.02) mRNA expression, CND was correlated with VEGF (R = 0.53, P < 0.002), ANGPT2 (R = 0.45, P < 0.008), FGF2 (R = 0.33, P < 0.05) and HIF1A (R = –0.48, P < 0.004), CSD was correlated with VEGF (R = 0.46, P < 0.007), FLT1 (R = 0.42, P < 0.01), ANGPT1 (R = 0.36, P < 0.03), ANGPT2 (R = 0.61, P < 0.002), FGF2 (R = 0.51, P < 0.002), and HIF1A (R = –0.39, P < 0.02) mRNA expression, and APC was correlated with VEGF (R = –0.40, P < 0.02), ANGPT1 (R = –0.33, P < 0.05), ANGPT2 (R = –0.33, P < 0.05), NOS3 (R = 0.32, P < 0.06), and HIF1A (R = –0.53, P < 0.001) mRNA expression.


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  		TABLE 2. Correlation of measures of angiogenesis with the expression of mRNA for the major angiogenic factors in the maternal placental (CAR) portion of the sheep placenta throughout the last two thirds of gestation.*


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  		TABLE 3. Correlation of measures of angiogenesis with the expression of mRNA for the major angiogenic factors in the fetal placental (COT) portion of the sheep placenta throughout the last two thirds of gestation.*

DISCUSSION

The formation of new vessels occurs via vasculogenesis and angiogenesis; both of these processes are regulated by a number of promoters and inhibitors [11, 12, 29] and are crucial for placental formation and the maintenance of pregnancy. Vasculogenesis is defined as de novo differentiation of endothelial cells from mesodermally derived precursors called angioblasts, or hemangioblasts, which are thought to represent unorganized, embryonic endothelial cells. Once formed, the angioblasts begin to cluster and reorganize to form capillary-like tubes [30]. Once this primary embryonic vascular network is formed, new capillaries are thought to form exclusively by the process of angiogenesis, which involves sprouting or splitting [31] from preexisting capillaries or the immediate post-capillary venules [32, 33].

In the present study, we evaluated primarily angiogenesis rather than vasculogenesis, as our observations were made during last two thirds of pregnancy (i.e., beginning on Day 50 of gestation). However, the fetal COT appears to be a relatively poorly vascularized tissue until the last half of gestation, and until this time, the cores of the fetal villi are composed of primarily amorphous connective tissue, sometimes referred to as Wharton jelly [7, 8]. After midgestation, the cores of the fetal villi, which comprise the distal ends of the COT, undergo dramatic cellular proliferation and tissue remodeling, which results in extensive vascularization [7–9]. Thus, vasculogenesis may still be occurring in this rapidly growing placental tissue even during the latter half of gestation. Nevertheless, it seems reasonable to suggest that most of the vascularization that occurs in the COT during the last two thirds or latter half of gestation involves sprouting and non-sprouting angiogenic processes. The latter would also encompass processes involved in the vascularization of the CAR, since the vessels in adults undergo angiogenesis exclusively.

The sheep chorioallantoic placenta makes an ideal model to study placental development, as the maternal (CAR) and fetal (COT) portions of the placenta remain closely associated but relatively intact throughout gestation, and each tissue can be evaluated separately [2, 3, 17]. The differences in vascular architecture and expression of mRNA for angiogenic factors in CAR and COT throughout normal pregnancy in sheep suggest differences in the regulation of their growth as well as their physiological functions.

Vascular endothelial growth factor is critical for vascular formation and is required in the initiation and formation of blood vessels by both vasculogenesis and angiogenesis [13, 14]. VEGF induces endothelial cell proliferation and promotes cell migration. In vivo, VEGF induces permeability of blood vessels and promotes vascular endothelial cell proliferation [34, 35]. Disruption of both VEGF alleles in mice results in poor vascular development, and disruption of even a single allele leads to embryonic lethality due to severe vascular abnormalities in the fetus and placenta [13, 14].

In the present study, we observed increases in VEGF mRNA expression from Day 50 through Day 110 of gestation for both COT and CAR. These increases follow closely the patterns of uterine and umbilical blood flow during pregnancy [3]. Interestingly, all of the measures of vascularity that we report in the present study are correlated with VEGF mRNA expression in COT but not in CAR. It appears that the concentration of VEGF mRNA, which is a pivotal mediator of angiogenesis, is higher on the fetal side than on the maternal side throughout last two thirds of gestation (Fig. 5), which may account for the higher rate of vascular growth that we observed in the COT compared with the CAR.

VEGF binds to its tyrosine kinase receptors, FLT1 and KDR. KDR seems to mediate the major growth and permeability effects of VEGF, whereas FLT1 may have a negative role, either by acting as a decoy receptor or by suppressing signaling through KDR [36]. However, within this conceptual framework, the different patterns of expression of VEGF receptors in CAR and COT do little to explain the highly branched pattern of angiogenesis in the fetal placenta compared with the dilation-like pattern of angiogenesis in the maternal placenta.

VEGF must work in concert with other factors to regulate angiogenesis, and it seems that angiopoietins play an important role in this regulation [37]. Both ANGPT1 and ANGPT2 bind primarily to the same TEK receptor with similar affinities. ANGPT1 is required for remodeling, stabilization, and maturation of developing vessels. ANGPT1 phosphorylates tyrosine in TEK, is chemotactic for endothelial cells, induces sprouting, and potentiates VEGF actions, although it fails to induce endothelial proliferation [37, 38]. ANGPT1 stabilizes networks initiated by VEGF, presumably by stimulating the interaction between endothelial cells and pericytes [37, 38]. Physiologically, ANGPT1 promotes angiogenesis by inducing vessel maturation and stabilization [39] and, in combination with VEGF, increases luminal diameter. The higher expression level of mRNA for ANGPT1 in the fetal compared with the maternal placenta may account for the development of the very dense and highly branched capillary network in COT by the end of pregnancy.

Nitric oxide is an intercellular messenger and is a critical local modulator of blood flow [40, 41]. Endothelial nitric oxide synthase is found in endothelial cells and produces NO, resulting in the dilatation of blood vessels. NO activates the enzyme GUCY1B3, which catalyses the conversion of guanosine 5'-triphospahte (GTP) to guanosine 3',5'-monophosphate (cGMP). This pathway is the mechanism by which NO regulates smooth muscle tone and thus, local blood flow [40, 41]. The primary change in the NO system observed in the present study was an increase in GUCY1B3 mRNA in CAR by Day 140.

Basic FGF appears to be one of the most widely distributed growth factors [11]. Many cells store FGF2 in a biologically inactive form, and the release of stored FGF2 may be a way to rapidly mobilize it for the stimulation of angiogenesis and cell proliferation. Basic FGF is a mitogenic and chemotactic factor for endothelial cells and it also stimulates endothelial production of collagenase and plasminogen activator proteases for degradation of the capillary basement membranes leading to endothelial cell migration during an angiogenic event [11]. In vitro experiments have shown that FGF2 signaling contributes to multiple steps of vessel formation, such as hemangioblast differentiation, endothelial cell migration and proliferation, and capillary assembly during vasculogenesis [42, 43] and sprouting angiogenesis [44, 45]. Basic FGF and FGFR gene knockout models in mice cause arrest of development before the onset of vascularization. In the present study, FGF2 expression changed little in the maternal (CAR) portion but increased exponentially in the fetal portion (COT) of the placenta; this observation indicates that FGF2 is primarily a fetal angiogenic factor or more likely, together with the other angiogenic factors, such as VEGF and ANGPT, it is important for the branching morphogenesis observed in the COT tissues.

Hypoxia-inducible transcription factor {alpha} (HIF1A) is a single regulatory protein that triggers a coordinated response of angiogenesis and arteriogenesis by inducing the expression of VEGF, FLT1, KDR, ANGPT2, Tie-1, NOS3, and many more factors [46, 47]. These effects have been observed in HIF1A- and VEGF-deficient embryos [13, 14]. Hypoxia is an important stimulus for the expansion of vascular bed, as cells are originally oxygenated by simple diffusion of oxygen, which is limited. We observed elevated levels of HIF1A mRNA expression in CAR and COT tissues from Day 50 till Day 110, at which time blood vessels start to grow more rapidly, which indicates a role for HIF1A in placental angiogenesis in sheep.

The observations made in the present study show different patterns of blood vessel growth and remodeling in CAR and COT (Figs. 3 and 4). These were accompanied by differences in the concentration and the pattern of expression of mRNA for angiogenic factors. Our histomorphometric study shows that the CAR capillary beds grow primarily by a 2.2-fold increase in the size of capillaries, which we define as APC, with only a small (1.5-fold) increase in capillary number. This dilation-like vessel growth can be explained by the correlation of CAD with GUCY1B3 along with ANGPT2 and FLT1 mRNA expression. The outcome of these relatively small increases in both APC and CND is a 3.3-fold increase in CAD in the CAR. This results in a small (1.7-fold) increase in CSD, which is an important measure of maternal placental physiological exchange. These observations indicate that the role of CAR is primarily to deliver nutrients via a low-velocity, irrigation-like flow [17]. This agrees with observations made for other species. For example, in the hemochorial placenta of primates, the maternal endometrium is lost and the fetal villi are bathed in a pool of maternal blood [2].

In contrast, the COT capillary beds grew primarily by branching and sprouting, resulting in a large (12-fold) increase in CND. In addition, we observed a decrease in capillary size (1.9-fold) accompanied by a 6.2-fold increase in CAD. This type of growth resulted in a large (6-fold) increase in the surface density of the fetal placental capillaries, which is the primary area of nutrient, respiratory gas, and waste exchange. This system seems to be ideal for nutrient uptake and delivery to the fetus [17], and this type of angiogenesis primarily correlates with the levels of mRNA expression for several angiogenic factors.

Adequate blood flow to the placenta is critical for normal fetal growth, which is confirmed by the observation that intrauterine growth restriction (IUGR) in third trimester human pregnancies is characterized by impaired uterine (maternal placental) and umbilical (fetal placental) blood flows, leading to reduced fetal nutrient uptake [48]. Reduced uterine blood flow during pregnancy can also be used as a predictor of high-risk pregnancies [49, 50]. Numerous studies indicate that increased blood flow rather than increased extraction rate is the primary mechanism of increased transplacental exchange, at least in a normal pregnancy [6, 10]. It has been suggested that angiogenic and vasoactive factors might serve as therapeutic targets in compromised pregnancies [51–53].

The different patterns of expression of mRNA for angiogenic factors, along with the different patterns of vascular growth in the fetal and maternal placental tissues can be useful in explaining the molecular mechanisms of angiogenesis in these tissues. These mechanisms are likely to include significant interactions among the various factors, such as those we have suggested for VEGF and ANGPT1, although the present study was not designed to investigate these interactions. Similarly, it seems likely that angiogenic factors from one of the placental tissues (i.e., CAR or COT) affect angiogenesis in the other tissue because of their close apposition, a possibility which, again, was not examined in the present study. Nonetheless, identifying the major angiogenic factors and evaluating their expression levels and correlating them with measurements of vascularity allow one to model angiogenesis in a three-dimensional, spatiotemporal fashion. In the future, we plan to use these data to develop mathematically rigorous three-dimensional models of fetal and maternal placental vascular growth throughout pregnancy, in both normal pregnancies and compromised pregnancies (i.e., pregnancies in which fetal growth is reduced).

FOOTNOTES

1Supported by National Institutes of Health grant HL64141 to L.P.R. and D.A.R. Back

Correspondence: 2Lawrence P. Reynolds, Center for Nutrition and Pregnancy, and Department of Animal and Range Sciences, North Dakota State University, Fargo ND 58105-5727. FAX: 701 231 7590; e-mail: Larry.Reynolds{at}ndsu.edu

Received: 14 June 2006.

First decision: 14 July 2006.

Accepted: 17 October 2006.

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