Placental angiogenesis in sheep models of compromised pregnancy

Abstract

Because the placenta is the organ that transports nutrients, respiratory gases and wastes between the maternal and fetal systems, development of its vascular beds is essential to normal placental function, and thus in supporting normal fetal growth. Compromised fetal growth and development have adverse health consequences during the neonatal period and throughout adult life. To establish the role of placental angiogenesis in compromised pregnancies, we first evaluated the pattern of placental angiogenesis and expression of angiogenic factors throughout normal pregnancy. In addition, we and others have established a variety of sheep models to evaluate the effects on fetal growth of various factors including maternal nutrient excess or deprivation and specific nutrients, maternal age, maternal and fetal genotype, increased numbers of fetuses, environmental thermal stress, and high altitude (hypobaric) conditions. Although placental angiogenesis is altered in each of these models in which fetal growth is adversely affected, the specific effect on placental angiogenesis depends on the type of 'stress' to which the pregnancy is subjected, and also differs between the fetal and maternal systems and between genotypes. We believe that the models of compromised pregnancy and the methods described in this review will enable us to develop a much better understanding of the mechanisms responsible for alterations in placental vascular development.

Figure 1. Schematic representation of the sheep placentome

The maternal, or caruncular, portion is represented by the stippled areas, and the fetal, or cotyledonary, portion is represented by the greyish areas. The vascular supply for each portion of the placentome is represented by the red (maternal, caruncular) or yellow (fetal, cotyledonary) vessels. Figure adapted from The Placenta: Human and Animal, Ramsey, Elizabeth Mapelsden. Copyright © (1982) by Praeger Publishers. Reproduced with Permission of Greenwood Publishing Group, Inc., Westfoot, CT.

Figure 2. Vascular development of maternal caruncle (CAR) and fetal cotyledon (COT) throughout the last two-thirds of gestation in sheep

Capillary area density (capillary area as a proportion of total tissue area) of both maternal (caruncle) and fetal (cotyledon) placental tissues increased exponentially from day 50 through day 140 after mating. Capillary area density is a commonly used measure of angiogenesis, and based on stereological principles also is equivalent to capillary volume density (capillary volume as a proportion of total tissue volume; Weibel, 1972; Hudlicka, 1984). Figure taken from Reynolds et al. (2005b).

Figure 3. Empirical model of angiogenesis in the sheep placenta throughout the last two-thirds of gestation

The maternal (caruncular) capillary beds grow primarily by increased capillary size, with only small increases in capillary number or surface densities, resulting in a modest increase in capillary area density. In contrast, the fetal (cotyledonary) capillary beds grow primarily by branching, resulting in a large increase in capillary number density accompanied by a decrease in capillary size; this branching pattern of growth also explains the relatively large increase in capillary area and surface densities. For illustration the capillary beds are depicted as open-ended, rather than as a closed loop. Taken from Reynolds et al. (2005b).

Figure 4. Angiogenic factor mRNA expression in placentomes (maternal caruncular + fetal cotyledonary tissue) from adolescent ewes on day 80 of pregnancy

Ewes were individually offered either a Moderate or High level of a diet calculated to promote moderate or rapid maternal growth rates, respectively. mRNA expression was quantified by real-time RT-PCR. Adapted from Redmer et al. (2005); *indicates effect of diet (P < 0.005), ns = no significant effect of diet.

Figure 5. Effects of nutrient restriction on placental expression of VEGF and VEGFR mRNAs

In this study, adult ewes received restricted (Restrict., 60% of NRC requirements) or maintenance (Maint.) diets from day 50 until day 90 (D 90) or day 130 (D 130) of pregnancy (length of gestation approximately 145 days). mRNA expression was quantified by ribonuclease protection assay. †Day effect (P < 0.01) for VEGF, VEGFR-1, and VEGFR-2. ‡Diet effect (P < 0.01) for VEGFR-1 and VEGFR-2. n = 5 ewes per group; s.e.m.= 193.7 for VEGF, 312.0 for VEGFR-1, and 203.1 for VEGFR-2. Taken from Redmer et al. (2004b).

Figure 6. Impacts of elevated maternal dietary selenium on fetal (A) and placental (B) growth in ewes

Ewes (n = 8–10 per group) were fed diets with similar nitrogen and energy contents but with normal levels of Se (Control, 0.1 p.p.m), or 3 p.p.m. of Se from an organic (Se, Wheat) or inorganic (Se Salt, 3 p.p.m) source, or 15 p.p.m. of Se from an inorganic source (Se Salt, 15 p.p.m.), beginning on day 50 after mating and continuing until slaughter on day 130. Although fetal weights (A) did not differ among groups, placental weight (B) was less (P < 0.02) in Se-treated versus Control ewes.

Figure 7

Impacts of elevated maternal dietary selenium on caruncular cell proliferation (A) (BrdU labelling index) and cotyledonary capillary number density (B) in ewes receiving diets containing Normal (0.1 p.p.m) or High (3 p.p.m. from high-Se wheat) levels of Se from day 50 until slaughter on day 130 of gestation. Borowicz, Ward, Vonnahme, Caton, Redmer & Reynolds, unpublished data.

Figure 8

Fetal weight, caruncular (CAR; maternal placental) weight, and cotyledonary (COT; fetal placental) weight in Romanov and Columbia lambs (Lamb; peripubertal) and yearlings (Yrlg; early adult) on day 130 of gestation. n = 5–7 per group; Lamb versus Yearling, P < 0.006 for fetal weight, P < 0.05 for total CAR weight, and P < 0.03 for total COT weight.

Figure 9

Expression of angiogenic factors in caruncular (CAR; maternal placental) and cotyledonary (COT; fetal placental) tissues of Romanov and Columbia lambs (Lamb; peripubertal) and yearlings (Yrlg; early adult) on day 130 of gestation. n = 7–12 per group; Lamb versus Yearling, P < 0.05 for VEGF and ANG-1 for COT only.

Figure 10

Fetal number, weight per fetus, total fetal weight, and caruncular and cotyledonary capillary area density (vascular density) on day 130 after mating in straight-bred Romanov (R × R) and Columbia (C × C) pregnancies or when Romanov embryos were transferred to Columbia dams (R → C). n = 4–9 per group. Bars with different superscripts differ, P < 0.04 for fetal no., P < 0.10 for fetal wt, P < 0.01 for vascular density.

Figure 11

Fetal and cotyledonary (COT) weights, cotyledonary capillary area density and total capillary volume, and correlation between cotyledonary total capillary volume and fetal weight on day 130 of gestation in mature ewes bearing singles, twins, or triplets. n = 15, 12, and 3 ewes per group, respectively.