The relationship between transplacental O2 diffusion and placental expression of PlGF, VEGF and their receptors in a placental insufficiency model of fetal growth restriction
Timothy R.H. Regnault, et.al.
The Journal of Physiology (2003) v.550.2, pp. 641-656
Full text Article from The Journal of Physiology
Abstract
Placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) are involved in placental angiogenesis through interactions with the VEGFR-1 and VEGFR-2 receptors. The placenta of pregnancies whose outcome is fetal growth restriction (FGR) are characterized by abnormal angiogenic development, classically associated with hypoxia. The present study evaluated the near-term expression of this growth factor family in an ovine model of placental insufficiency-FGR, in relationship to uteroplacental oxygenation.
Compared to controls, FGR pregnancies demonstrated a 37 % increase in uterine blood flow (FGR vs. control, which was associated with an increased maternal uterine venous, increased umbilical artery systolic/diastolic ratio, and fetal hypoxia. Maternal caruncle PlGF mRNA was increased in FGR, while fetal cotyledon VEGF mRNA was reduced.
The data establish that uterine blood flow is not reduced in relationship to metabolic demands in this FGR model and that the transplacental PO2 gradient is increased, maintaining umbilical oxygen uptake per unit of tissue. Furthermore, these data suggest that an increased transplacental gradient of oxygen generates changes in angiogenic growth factors, which may underline the pathophysiology of the post-placental hypoxic FGR.
RESULTS
Table 1 presents mean CBT, breathing rate, and feed and water intakes of all ewes prior to removal of the HT group from the environmental chamber at approximately 120 dGA and again at 135 dGA under control TN conditions, on the day of study and/or tissue collection. The CBT of ewes in the HT treatment was elevated at 120 dGA The breathing rate of ewes in the HT treatment was increased 8-fold while in treatment, and this reverted to values comparable to those of TN ewes once the ewes were housed in TN conditions. Feed and water intakes were constant between the treatment groups and within treatment groups across time, both prior to HT removal and at study and/or tissue collection.
Placental and fetal tissue weight comparisons and placental resistance indices
Compared to the control (TN) animals, the HT-exposed animals demonstrated significant reductions in both placental and fetal weights. These reductions were equivalent to a 50 % reduction in placental weight and a 40 % reduction in fetal weight. The brain/liver weight ratio was increased in HT fetuses, while the ponderal index was not different between the two groups.
Figure 1 presents the umbilical artery resistance indices. All three calculations of resistance were increased in HT pregnancies, Umb S/D 1.8-fold (P < 0.005), Umb PI 1.9- fold (P < 0.01) and Umb RI 1.6-fold (P < 0.005). These increased values are indicative of increased resistance to blood flow downstream from the sampling site in the umbilical artery, within the placental vascular bed. 
Figure 1 :
Umbilical arterial systolic/diastolic (Umb S/D), umbilical placental resistance (Umb PI) and umbilical resistance index (Umb RI) in TN and HT pregnancies, determined following HT treatment.
Uterine and umbilical blood flows, ethanol clearance and fetal and uteroplacental oxygenation
Uterine blood flows were all significantly increased when expressed per kg conceptus, per 100 g placenta or per kg fetus in HT-exposed ewes (Table 3). Umbilical blood flow (ml min-1) was reduced in the HT group and umbilical blood flow per 100 g placenta was also depressed. The uterine/umbilical blood flow ratio was increased approximately 2-fold.
Maternal uterine vein oxygen content and saturation were significantly greater in HT than TN ewes. Uterine arteriovenous difference for oxygen content was greater in the TN pregnancies, whereas maternal haematocrit was similar in both groups. HT umbilical vein and fetal pedal artery displayed increased oxygen capacity levels, associated with an increased haematocrit (Table 4). Fetal arterial and umbilical venous PO2 were reduced in HT pregnancies.

The transplacental PO2 and PCO2 gradients were calculated as the difference between uterine vein and umbilical vein PO2 and PCO2, respectively (Table 5). Though the PCO2 gradient was not affected by treatment, the PO2 gradient was significantly increased in the HT pregnancies. Uterine and umbilical oxygen uptakes were decreased in HT pregnancies. The percentage uterine oxygen extraction was decreased in HT pregnancies, a consequence of a high rate of flow of maternal O2 to the pregnant uterus (i.e. arterial O2 content blood flow) in relation to uterine O2 uptake. However, oxygen uptake per unit weight was not different between TN and HT groups for uterine, umbilical or uteroplacental units (Table 5).

Comparison of uterine venous and umbilical venous PO2 and PCO2 differences in TN and HT pregnancies
Expression of PlGF, VEGF, VEGR-1 and VEGFR-2 in placenta tissues
A single major PlGF transcript of approximately 1.7 kb was expressed in both maternal caruncle and fetal cotyledon tissues at 135 dGA. The concentration of PlGF mRNA was greater in TN fetal cotyledon tissue than in caruncle tissue (P < 0.0008, Fig. 3A). The HT caruncle PlGF mRNA concentration was increased over that of the TN caruncle concentration (P < 0.02, Fig. 3A). In both placental tissues a major VEGF transcript of approximately 3.7 kb was expressed. Similar to PlGF mRNA tissue concentrations, the VEGF mRNA caruncle concentration was less than that observed in TN cotyledon tissue (P < 0.03, Fig. 3B). HT cotyledon tissues had a reduced concentration of VEGF mRNA compared to TN cotyledon tissues (P < 0.03, Fig. 3B). The VEGFR-1 mRNA concentration was greater in TN cotyledon tissues than in TN caruncle (P < 0.001, Fig. 4A), though in HT cotyledon tissues it was reduced 50 % (P < 0.0002, Fig. 4A). The concentration of VEGFR-2 mRNA was consistent across treatments within tissue type (Fig. 4B).
Figure 3
Concentration of ovine PlGF mRNA (A) and VEGF mRNA (B) (GF/actin mRNA densitometry units) in TN () and HT () maternal caruncle and fetal cotyledon tissues at 135 dGA.

Ovine PlGF (A) and VEGF (B) protein measured by Western immunoblot (GF/Actin) in TN and HT maternal caruncle and fetal cotyledon tissues.