Cecilia Y. Cheung

Vascular endothelial growth factor gene expression in ovine placenta and fetal membranes

OBJECTIVE The purpose of this study was to explore the gene expression of vascular endothelial growth factor (VEGF) in placental cotyledon, chorion, and amnion of the ovine fetus. STUDY DESIGN Time-dated pregnant sheep with singleton or twin fetuses at a gestational age ranging from 100 to 140 days were used for the study. Placental cotyledonary, chorionic, and amniotic tissues were collected and processed for messenger ribonucleic acid analysis by Northern blotting and reverse transcription-polymerase chain reaction. RESULTS By use of a phosphorus 32-labeled human VEGF complementary deoxyribonucleic acid probe, a prominent VEGF messenger ribonucleic acid transcript of 3.7 kb was detected in the cotyledon, chorion, and amnion. A minor band of 1.7 kb was also found but only in the cotyledon and chorion. The abundance of messenger ribonucleic acid encoding VEGF was highest (p < 0.001) in the cotyledon and lowest in the amnion. In these tissues polymerase chain reaction-amplified products corresponding to VEGF121, VEGF165, VEGF189, and VEGF206 were identified by ethidium bromide. In addition, a polymerase chain reaction fragment corresponding to VEGF145 was observed. These fragments produced specific hybridization signals with the human VEGF radioactive probe where the intensity of the signal was strongest for VEGF165 and weakest for VEGF189. CONCLUSIONS VEGF gene expression was detected in the cotyledon, chorion, and amnion of the near-term ovine fetus. These findings suggest that vascular endothelial growth factor may play a role in the induction of angiogenesis and promotion of permeability in the microvessels that perfuse the placental and fetal membranes.

Vascular Endothelial Growth Factor: Possible Role in Fetal Development and Placental Function

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen with potent permeability properties. This growth factor exists in several isoforms; the most abundant form present in most tissues is VEGF165. The different isoforms exhibit differences in biologic function. During development, VEGF is expressed in multiple embryonic and fetal tissues, with the highest levels found in the lung, kidney, and heart. Vascular endothelial growth factor is also expressed in placental tissues and fetal membranes, and this expression increases with advancing gestation. In the fetal heart and placenta, VEGF expression is inducible by hypoxia. Two receptors, KDR and Flt-1, have been identified for VEGF. They are widely expressed in vascular endothelial cells and are also found in placental tissues where VEGF is localized. In humans, Flt-1 appears to be the predominant receptor, whereas in the cow and sheep, KDR is the major receptor expressed. The presence of VEGF and its receptors in placental tissues throughout gestation strongly suggests that VEGF plays an important role in the development and maintenance of placental vascular function during pregnancy. The localization of VEGF in fetal membranes and the fetal surface of the placenta raises the possibility that VEGF may be involved in the regulation of amniotic fluid volume and composition.

Rapid intramembranous absorption into the fetal circulation of arginine vasopressin injected intraamniotically

Recently an intramembranous pathway was reported in the ovine fetus as a route for the movement of a significant volume of water from the amniotic cavity directly into the fetal blood, which perfuses the fetal membranes and fetal surface of the placenta. To test whether this pathway could be an avenue for the movement of arginine vasopressin from the amniotic cavity into the fetal circulation, we injected 1 to 25 micrograms of arginine vasopressin into the amniotic cavity of two groups of chronically catheterized fetal sheep: a control group of seven animals and a group of seven animals with surgical ligation of the fetal esophagus. We found similar and highly significant increases of arginine vasopressin concentrations in both control and surgically ligated fetuses in amniotic fluid (p less than 0.00001), fetal plasma (p less than 0.0001), and fetal urine (p less than 0.0001). Both groups had similar increases in arterial (p less than 0.0001) and venous (p less than 0.003) pressures with simultaneous decreases in urine flow (p less than 0.001) and heart rate (p less than 0.0001) after the intraamniotic injection of arginine vasopressin. We conclude that amniotic arginine vasopressin can be rapidly absorbed in its biologically active form directly into the fetal circulation through the intramembranous pathway. Furthermore, the observation that esophageal ligation did not alter this absorption suggests that the intramembranous pathway may be important in the regulation of amniotic fluid volume and composition.

Fetal hypoxia elevates plasma atrial natriuretic factor concentration

Acute hypoxia in the fetus is associated with a reduction in fetal blood volume. We hypothesized that atrial natriuretic factor in the fetal circulation may be one of the factors that mediate this blood volume decrease. Thus the present study was designed to determine the effects of hypoxia on circulating concentrations of atrial natriuretic factor in fetal sheep and correlate these changes with fetal blood volume. Hypoxia was induced in chronically catheterized sheep fetuses by infusing nitrogen containing CO2 into the trachea of the ewe for 30 minutes. Fetal arterial PO2 decreased by 10.2 +/- 1.3 (SE) mm Hg. Plasma atrial natriuretic factor concentration rose concurrently with the fall in PO2 such that atrial natriuretic factor increased to 565 +/- 196 pg/ml from a basal level of 127 +/- 13 pg/ml (p less than 0.001). Fetal blood volume was reduced by 7.2% +/- 2.1% and was significantly related to changes in atrial natriuretic factor levels (p less than 0.0001). At the termination of hypoxia, PO2 returned to normal levels before plasma concentrations of atrial natriuretic factor fell to baseline values. Therefore fetal hypoxia appears to be a potent stimulus for elevating plasma concentration of atrial natriuretic factor in the fetus, and this rise in atrial natriuretic factor in the circulation may be partially responsible for the reduction in fetal blood volume observed during hypoxia.

Vascular endothelial growth factor activation of intramembranous absorption: A critical pathway for amniotic fluid volume regulation

Objective: The purpose of this review is to propose a critical role for vascular endothelial growth factor (VEGF) in mediating the transfer of amniotic fluid from the amniotic compartment through the fetal membranes and fetal surface of the placenta into fetal blood. Methods: Experimental findings in humans and animal models on the action of VEGF in mediating fluid transfer are reviewed and interpreted in order to postulate a proposed mechanism for VEGF regulation of amniotic fluid absorption through the fetal membranes and placenta. Results: Recent scientific advances suggest that up-regulation of VEGF gene expression in the amnion and choiron is associated with increased transfer of amniotic fluid into fetal blood. The possible mechanisms of action for VEGF appear to involve regulation of intramembranous blood vessel proliferation and membrane transport via passive permeation as well as nonpassive transcytotic vesicular movement of fluid. Conclusion: Currently evolving concepts suggest that amniotic fluid volume is regulated through modulation of the rate of intramembranous absorption of amniotic fluid by both passive and nonpassive mechanisms. The permediability factor VEGF appears to be a critical regulator of amniotic fluid trapsort in the fetal membranes.

Developmental expression of vascular endothelial growth factor and its receptors in ovine placenta and fetal membranes.

Objectives: Vascularity of the surface of the placenta in humans and of the placenta and fetal membranes in several species including sheep is an important determinant of intramembranous absorption of amniotic fluid. Our previous studies have shown that the total blood vessel surface area in ovine amnion and chorion increases with advancing gestation. Vascular endothelial growth factor (VEGF) is a potent angiogenic and pemeability factor and is found to be expressed in the ovine placenta and fetal membranes. To investigate the role of VEGF in maintaining the absorptive function of the intramembranous microvessels, the present study was undertaken to determine the gestational change in gene expression of VEGF and its receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1), in ovine placenta, chorion, and amnion. Methods: Total RNA was extracted from placental cotyledon, chorion, and amnion of ovine fetuses at 60-140 days of gestation. The relative abundance of VEGF, KDR, and Flt-1 mRNA was determined by Northern blot analysis, and VEGF molecular forms expressed were identified by reverse transcriptase polymerase chain reaction. The gestational changes in mRNA levels of VEGF and its receptors were analyzed by regression analysis. Results: In ovine placenta, chorion, and amnion, VEGF mRNA levels increased significantly from 60 to 140 days. The major VEGF molecular form expressed in these tissues was VEGF164, whereas VEGF120, VEGF144, and VEGF188 were present at lower levels. In the placenta, KDR was the primary VEGF receptor expressed, although Flt-1 was also detected at very low levels. In the amnion and chorion, KDR was the only receptor expressed. A gestational-dependent change in VEGF receptor expression was not observed in the placenta and membranes. Conclusions: The increase in VEGF gene expression with advancing gestation in the amnion and chorion where KDR is expressed suggests that VEGF and its receptor are important determinants of vascularity and permeability, and thus exchange capacity, of the intramembranous pathway.

Ovine Vascular Endothelial Growth Factor: Nucleotide Sequence and Expression in Fetal Tissues

To examine the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during fetal development, we determined the gene expression of VEGF in ovine fetal tissues. Further, we cloned and sequenced the ovine VEGF cDNA encoding VEGF164 from sheep placenta. VEGF protein was localized in epithelial cells of the placenta and fetal kidney, and in hepatocytes of the fetal liver. By Northern analysis, a major VEGF mRNA species of 3.7 kb was identified in all tissues examined, with abundance highest in the lung and lowest in the liver. The most prominent molecular form expressed in ovine fetal tissues appeared to be VEGF164 with low levels of expression of VEGF120 and VEGF188. Cloning and sequence analysis of the most abundant form of ovine VEGF cDNA in the placenta confirmed the prediction of a 164-amino acid peptide, with a putative N-terminal signal sequence of 26 amino acids. Comparison of the VEGF cDNA sequence among different species revealed that VEGF is highly conserved suggesting an important role in development.

Regulation of Amniotic Fluid Volume: Evolving Concepts

Studies in late gestation fetal sheep have provided several new insights into the regulation of amniotic fluid (AF) volume (AFV): There are four quantitatively important amniotic inflows and outflows that include fetal urine production, lung liquid secretion, swallowing, and intramembranous absorption. Of these, AFV is regulated primarily by modulating the rate of intramembranous absorption of AF water and solutes across the amniotic epithelial cells into the underlying fetal vasculature. Modulation of the rate of intramembranous absorption depends on the presence of stimulators and inhibitors present in the AF. A stimulator of intramembranous absorption is present in fetal urine. In addition, AF contains a non-renal, non-pulmonary inhibitor of intramembranous absorption presumably secreted by the fetal membranes. Although passive bidirectional movements of water and solutes occur across the intramembranous pathway, intramembranous absorption is primarily a unidirectional, vesicular, bulk transport process mediated through VEGF activation of transcytotic transport via caveolae. Further, the stimulators and inhibitors of intramembranous absorption alter only the active, unidirectional component of intramembranous absorption while the passive components are not altered under experimental conditions studied thus far. Future progress depends on identifying the cellular and molecular mechanisms that regulate active and passive intramembranous absorption as well as their regulatory components.

Amniotic Fluid Volume and Composition in Mouse Pregnancy

Objective: The current study was undertaken to determine simultaneous changes in amniotic fluid (AF) volume and composition across gestation in the pregnant mouse. Methods: Young adult mice (6 to 7 weeks old) of the CB6F1 strain were mated overnight. AF was collected on consecutive days from embryonic days 9.5 through 18.5 for measurements of volume and composition. Statistical analysis included one-factor analysis of variance (ANOVA). Results: Af volume increased from 18 ± 4 (Se) μL on day 9.5 to a maximum of 147 ± 4 μL on days 15.5 to 16.5 and decreased sharply to 17 ± 3 μL on day 18.5 AF osmolality was unchanged except for a rise prior to delivery on day 19.5 to 20.5 AF sodium, calcium, and glucose concentrations increased and subsequently decreased as gestation progressed. AF potassium, chloride, and loctate concentrations initially decreased and then increased across gestation. Prior to day 9.5 and after day 18.5, AF volume was too small for volume or compositional determinations. Conclusions: In the mouse, the rise in AF volume from mid gestation to a maximum late in gestation is similar to that in humans while the sharp fall prior to delivery is not. As observed in the fetal sheep, the changes in fluid volume are associated with aF osmolality and solute concentration changes that are correlated with advancing gestational age. These observations together with the feasibliity of quantifying AF volume and composition in the mouse fetus demonstate the possibility of using genetically altered mice as a model for future studies on the molecular mechanisms underlying the regulation of AE volume and composition.

Indomethacin-induced urinary flow rate reduction in the ovine fetus is associated with reduced free water clearance and elevated plasma arginine vasopressin levels

OBJECTIVE The purpose of our study was to explore the urinary responses of the ovine fetus to indomethacin levels comparable with those used therapeutically in the human fetus. STUDY DESIGN After a 1-hour control period, chronically catheterized ovine fetuses between 125 and 139 days of gestation were given an intravenous bolus of indomethacin (0.05 mg/kg estimated fetal weight) followed by a 0.0025 mg/kg/min continuous infusion for 5 hours. The experimental group (n = 9) was compared with a vehicle-only infusion group (n = 10). RESULTS There was a sustained 55.7% +/- 9.5% (mean +/- SEM) decrease in urinary output by 2 hours of indomethacin infusion (p < 0.00001, analysis of variance). Urinary osmolality, potassium, and chloride concentrations underwent sustained increases during the infusion period (p < 0.005). Free water clearance decreased by 67.5% +/- 12.0% (p < 0.001). Fetal arterial pressure increased only transiently (p < 0.05), and increases in venous pressure (p = 0.013) and heart rate (p < 0.0001) were sustained. Fetal plasma arginine vasopressin concentration increased during indomethacin infusion (p < 0.05) and was correlated with the fall in urinary flow rate and free water clearance (p = 0.002). During vehicle infusion no significant changes were observed in any of the variables. CONCLUSIONS Our data indicate that the fetus undergoes antidiuresis when exposed to low levels of indomethacin and that the observed antidiuresis is mediated by a decrease in free water clearance. The reduction in free water clearance may be mediated by increases in plasma arginine vasopressin concentrations.