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Regular Article |
a Perinatal Research Laboratories, Departments of Obstetrics/Gynecology and
b Animal Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53715
ABSTRACT
During the third trimester, fetoplacental and uterine blood flows increase dramatically to meet the high metabolic demands of the growing fetus. We hypothesized that the expression of endothelial nitric oxide synthase (eNOS) in fetoplacental artery endothelium and the concentrations of nitric oxide (NO) and cyclic GMP (cGMP) in amniotic fluid (AF) are increased during the third trimester of ovine gestation. Placental arteries and AF were collected from ewes at 110, 120, 130, and 142 days of gestation (n = 24; mean ± SEM term = 145 ± 3 days). Expression of eNOS protein was measured in intact and denuded placental arteries and in endothelium-derived protein by Western analysis and confirmed by immunohistochemistry. Concentrations of NO (nitrates plus nitrites) and cGMP were determined in AF. Placental artery eNOS protein expression was localized to the endothelium, where it was markedly greater than in vascular smooth muscle. Placental artery endothelium-derived eNOS expression and AF cGMP concentrations were similar at 110 and 120 days of gestation; however, both peaked at 130 days at levels two- to threefold above baseline (P < 0.05) before returning to baseline at 142 days of pregnancy. The AF NO (nitrates plus nitrites) levels, however, increased progressively between 120 days of gestation and term (P < 0.05). We concluded that endothelium-derived placental artery eNOS levels, AF NO (nitrates plus nitrites), and AF cGMP were markedly increased during the third trimester, thus supporting a role for NO-mediated elevations in cGMP in the control of fetoplacental blood flow.
cGMP, endothelium, nitric oxide, placenta, pregnancy
INTRODUCTION
During the third trimester of normal human and ovine gestation, rapid fetal growth and the increased metabolic demands of the growing fetus are accompanied by dramatic increases in fetoplacental and uteroplacental blood flows [1–3]. Elevations of placental blood flow in both the fetal and maternal compartments are also related directly to fetal development, fetal survival, and neonatal birthweights, which correspond to reductions in fetal/neonatal morbidity and mortality [1–3]. The increase in fetoplacental and uteroplacental blood flows, especially during the third trimester of pregnancy, results from both vasodilatation and neovascularization (angiogenesis) as the placenta continues to remodel [1, 2, 4, 5].
In the absence of autonomic innervation of the fetoplacental blood vessels, vascular relaxation occurs by the influence of circulating and locally produced vasodilators [6–9]. Local production of vasodilators by the vascular endothelium mediates some of the vasodilation that occurs in pregnancy [8–11]. One of the very potent but labile vasodilators produced by the fetoplacental artery endothelium [9] is nitric oxide (NO), which is synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Endothelium-derived NO locally relaxes the underlying vascular smooth muscle (VSM) by activating soluble guanylate cyclase and producing the second messenger cyclic GMP (cGMP), which reduces vasomotor tone [10, 12]. A physiologic cause-and-effect relationship between fetoplacental NO production and blood flow has previously been demonstrated from studies in which infusion of NOS inhibitors potentiated vasoconstriction of human stem villous placental arteries and decreased ovine fetoplacental blood flow [13, 14]. Although NO also is elevated in the maternal circulation during normal pregnancy in sheep and rats [9–11, 15], virtually nothing is known about its regulation in the ovine fetoplacental vasculature. Recently, we reported that uterine artery endothelium-derived (not VSM-derived) eNOS protein expression is increased in late pregnant versus nonpregnant sheep, and this increase was not observed in systemic (omental and renal) vasculature [11, 15]. Although the fetoplacental vascular endothelium has been shown to also express endothelium-derived eNOS [9, 16–21], the developmental changes in placental artery eNOS protein expression have not been described during the third trimester of gestation, a time of increased metabolic demand due to rapid fetal growth [1, 2, 9, 22, 23]. We therefore tested the hypotheses that 1) expression of eNOS increases in ovine fetoplacental artery endothelium during the third trimester and 2) this increase is associated with increases in amniotic fluid (AF) NO (measured as total nitrate plus nitrate [NOx]) and cGMP levels, the latter second messenger being the physiologic mediator of NO actions. The specific objectives of the current study were to demonstrate and compare the ontogeny of the cellular localization (endothelium and/or VSM) of eNOS isoform expression in fetoplacental arteries and to compare these changes with AF NOx and AF cGMP levels throughout the third trimester. We report herein for the first time that the eNOS protein expression in the fetoplacental artery endothelium is developmentally regulated and that increases in AF NOx and AF cGMP levels are also present during this time in the third trimester.
MATERIALS AND METHODS
Experimental Design
Polypay and mixed western breed multiparous pregnant ewes (55–75 kg) were killed at 110 (n = 5), 120 (n = 7), 130 (n = 7), 142 (n = 5) days of gestation (n = 24, mean ± SEM term = 145 ± 3 days) as previously described [8, 11, 12]. Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison Research Animal Care and Use Committee and followed the recommended Report of the American Veterinary Medical Association Panel on Euthanasia. Preoperatively, a catheter was introduced into the jugular vein and advanced into the right ventricle for central venous access. General anesthesia was induced with sodium pentobarbital (Nembutal; 25–50 mg/kg; Sigma Chemical Co., St. Louis, MO), and nonsurvival surgery was performed.
Procurement of Specimens
At laparotomy, the uterus was exteriorized. Through a small hysterotomy, the fetal membranes were exposed and an aliquot of AF was aspirated into a sterile syringe and frozen. The fetal lamb(s) was delivered and weighed, and crown-rump lengths were measured. After hysterectomy was performed, the animals were killed [8, 11, 12]. Placental arteries were identified at their origin, branching from the umbilical artery. First and second order fetoplacental arteries outside of the umbilical cord, with a diameter of 3–5 mm, were dissected from the membranes and thoroughly rinsed free of blood with PBS (10 mM PBS: 8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, pH 7.4).
Immunohistochemistry
Segments of second and third order placental artery (1–3 mm diameter) were fixed in 4% formaldehyde in sodium cacodylate buffer (0.1 M, pH 7.4), embedded in paraffin, and sectioned at 6 µm. After deparaffinization, the sections were incubated in 3% H2O2 in methanol for 15 min to quench endogenous peroxidase activity. Immunolocalization of eNOS was performed using isoform-specific antibodies (Transduction Laboratories, Lexington, KY) and indirect immunoperoxidase detection via the avidin-biotin-peroxidase system with 3,3' diaminobenzidine (ELITE ABC kit; Vector Laboratories, Burlingame, CA) as described previously [8, 11, 16]. Based on Western analysis data, fetoplacental artery sections from only three 110-day and three 130-day pregnant sheep were incubated with eNOS antibody (mouse monoclonal; 2.5 µg/ml in PBS containing 1% BSA) for 1 h at room temperature; the optimal concentration of primary antibodies had been determined in earlier studies [11, 16]. For the control specimens, the primary antibody was replaced with normal mouse IgG (Vector Laboratories) at 2.5 µg/ml. After immunostaining, tissue sections were briefly counterstained with Harris hematoxylin.
Preparation of Fetoplacental Arteries for Western Immunoblot Analysis
To quantitate the expression of eNOS as present mainly in the endothelium and not in VSM, we employed the rapid procedure to isolate the tunica intima containing endothelium-derived proteins, which we previously developed, described, and validated for uterine, omental, and renal arteries [11, 12]. Intact placental artery segments (3- to 5-mm diameter) were opened longitudinally, and the tunica intima was gently scraped from the vessel lumen. This isolated endothelium-derived protein preparation was transferred directly into lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 10 mM EDTA, pH 7.4, 0.1% Tween 20, 0.1% ß-mercaptoethanol, 0.1 mM phenylmethylsulfonylfluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin). Adjacent whole vessel "intact" artery segments (1–3 mm) also were placed directly into protein lysis buffer. The denuded vessel, consisting predominantly of VSM, was then rubbed with a wet cotton swab to remove any remaining endothelium, and any remaining adventia was extensively removed. The three placental artery-derived specimens, i.e., endothelium-derived preparations, intact arteries, and denuded arteries, were snap frozen in liquid nitrogen and stored at -20°C. In situations when sheep had twins or triplets, the endothelium-derived preparations were pooled so that there was only one sample per ewe.
Western Immunoblot Analysis
Intact and denuded arteries were homogenized in lysis buffer. Endothelium-derived proteins were sonicated in the same buffer. After centrifugation to remove insoluble material, the supernatants were assayed for protein content using a modified Lowry assay procedure (BioRad, Hercules, CA). Protein samples were separated by SDS-PAGE using human umbilical vein endothelial cell (HUVEC) protein (2.5 µg) as a positive control and an internal standard. Molecular weight "rainbow" standards (Amersham International, Arlington Heights, IL) were loaded onto one lane of each gel. Samples (30 µg of protein of intact or denuded vessel and 10 µg of endothelium-derived protein) were loaded onto the gel after 5 min of incubation at 92°C in a total volume of 25 µl, plus the sample buffer (final concentration: 0.05 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 2 mM EDTA). Proteins were separated on a 7.0% polyacrylamide gel with 0.1% SDS by electrophoresis at 100 V, incubated for 2 h at room temperature, and transferred onto Immobilon P (BioRad) membrane at 30 V overnight at 4°C using the Mini-Protean II system (BioRad). The membrane was probed by the Western technique, using eNOS subtype-specific antisera (mouse monoclonal, 1:750 dilution, 0.33 µg/ml; Transduction Laboratories), and the secondary antiserum was horseradish peroxidase-conjugated goat anti-mouse (Amersham International) at 1:1000 dilution. Proteins were detected with enhanced chemiluminescence reagents (Amersham International), and blots were exposed to Hyperfilm (Amersham International) for 1, 5, and 15 min. Signal intensities were quantified by scanning densitometry (Model 670 Scanning Densitometer; BioRad). The signal for the eNOS isoform corresponded to the HUVEC signal at an apparent molecular mass of 140–150 kDa. Densitometry data were normalized to the HUVEC signal and are reported as the mean percentage of HUVEC signal density.
Determination of NO (Nitrates Plus Nitrites) and cGMP in AF
Aliquots of AF were thawed, mixed, and centrifuged to remove debris. Levels of NO (nitrates plus nitrites) in AF were determined by chemiluminescence using an NO Analyzer (NOA 280; Sievers Instruments, Boulder, CO) [8, 15]. The concentration of cGMP was measured by ELISA (Cayman Chemical Co., Ann Arbor, MI) [11, 12], and results are reported as picomols of cGMP x 103 normalized to milligrams of AF creatinine (Sigma Chemical Co.).
Statistical Analysis
Data were analyzed by one-way analysis of variance and the Student unpaired t-tests assuming equal variance using Sigma Stat (Jandel Scientific, San Rafael, CA) and Excel (Microsoft, Redmond, WA) software. Significance was defined at P 
0.05. Data are reported as the mean ± SEM. Correlations among fetoplacental eNOS, AF cGMP, and AF NOx were calculated using the least squares methods and are reported as the r2 value for the highest order that significantly added to the model.
RESULTS
Fetal Measurements
Individual and total fetal weights and crown-rump length increased progressively over time during the third trimester, based on the four time points measured (Table 1). Mean individual fetal weight increased by 0.096 kg/day between 110 and 120 days, 0.17 kg/day between 120 and 130 days, and 0.051 kg/day between 130 and 142 days. Therefore, the maximum rate of individual fetal growth velocity (
weight/day) was observed after 120 days. Although total combined fetal weights per sheep also show similar patterns, four sheep in the 110-, 120-, and 142-day gestation groups had twins. Individual fetal weights are expected to be reduced for twins, thus reducing the maximal estimate of the rates of total fetal weight change per day observed between 120 and 130 days of gestation (0.09 kg/day). However, body mass index, derived from the individual fetal weight in kilograms divided by the square of the crown-rump length, was not substantially altered throughout the third trimester of gestation, confirming proportional fetal growth.
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Immunohistochemical Localization of eNOS
Intense immunostaining for eNOS was observed in the endothelium of intact placental arteries (Fig. 1). A very subtle, diffuse, and patchy pattern of staining for eNOS was also observed in the vascular wall in some but not all of the sections of both the antibody-exposed and control sections, suggesting nonspecific staining. No endothelial staining was observed in the negative control sections (mouse IgG) of the placental arteries studied. Qualitatively, there appeared to be somewhat greater immunostaining in Day 130 versus Day 110 gestation fetoplacental artery endothelium but not VSM.
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Endothelial Expression of eNOS by Western Immunoblot Analysis
Consistent with the qualitative immunohistochemical findings, eNOS was detected in the endothelium but not in the VSM of the fetoplacental arteries at all four gestational ages studied. We previously demonstrated the validity and necessity of separating endothelium and VSM for the quantitative evaluation of eNOS [11]. We detected eNOS levels by Western immunoblot analysis (Fig. 2) in both intact arteries and endothelium-derived protein preparations, but not in denuded arteries (mainly VSM). However, the intensity of the signal for eNOS was substantially greater in only 10 µg of endothelium-derived protein than in 30 µg of intact vessel homogenate. These data are consistent with the estimated enrichment of endothelium-isolated protein by this method, which was previously determined to be 700- to 800-fold for uterine arteries [11]. The intensity of the signal of the eNOS bands in both intact placental arteries and the endothelium-isolated proteins also was quantitatively greater at 130 than at 110 days of gestation (Fig. 2). As shown in a representative Western immunoblot (Fig. 3), preparations of endothelium-derived proteins showed a consistently more intense signal for eNOS at 130 days than at the other gestational ages studied. Signal intensity at 130 days, as a proportion of the same control HUVEC sample run on each blot, was 2- to 10-fold greater than at 110 days, with a mean ratio of nearly 2.5-fold (P = 0.01; Fig. 4). There was no significant difference in mean eNOS expression at 110, 120, and 142 days. Because multifetal gestations in sheep are associated with smaller fetuses, we also compared endothelium-derived eNOS protein expression in singleton versus twin/triplet pregnancies. We did not detect any differences (P = 0.67) in endothelial eNOS levels due to fetal number regardless of gestational age (singleton = 657 ± 169 AU/mg protein, n = 10; multiples = 753 ± 143, n = 14 AU/mg protein).
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Amniotic Fluid Concentrations of cGMP and NOx
The concentrations of cGMP in AF were unaltered from 110 to 120 days of gestation, peaked at 130 days (P = 0.03), and then returned to baseline at 142 days. This pattern of increase, peaking at 130 days at greater than three times the concentration of 110 days, exactly matched the pattern of eNOS increase (Fig. 4). As observed with the Western immunoblot data for the eNOS values, the AF cGMP at 130 days of gestation differed significantly from that observed on all other days (P < 0.03), whereas there was no significant difference between values at 110, 120, and 142 days. In contrast, levels of NOx in AF increased progressively (P < 0.05) throughout the third trimester of gestation (Fig. 5). As with eNOS levels, we did not detect any significant differences in either AF cGMP (2062 ± 1228 and 957 ± 304 pmol/mg, respectively; P = 0.26) or AF NOx (319 ± 36 and 497 ± 67 µM, respectively; P = 0.06) levels in singleton (n = 10) versus multifetal (n = 14) gestations.
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Correlative Relationships among eNOS, NOx, and cGMP
Placental artery endothelial eNOS expression was highly correlated with AF cGMP (r2 = 0.78; linear) concentrations. In addition, AF NOx levels were moderately correlated with AF cGMP levels (r2 = 0.27; cubic).
DISCUSSION
In the present study we quantitatively showed on Western immunoblots that the VSM eNOS staining was not evident, although eNOS levels in both intact placental arteries and endothelium-derived protein were prominent. These data are consistent with our previous observations that eNOS expression is predominantly localized to the endothelium and not the VSM of maternal (uterine, omental, and renal) arteries during the third trimester of ovine pregnancy [11]. Positive immunohistochemical staining of fetoplacental artery eNOS during the third trimester in the sheep is also in agreement with the abundant eNOS expression in the human placental stem villous vasculature [9, 17, 19, 21].
Using Western analysis we then demonstrated for the first time that the expression of eNOS in the endothelium increased dramatically by 130 days, peaking at levels two to three times higher than the earlier gestational ages tested (110 and 120 days) before declining at term, coincident with the changes in AF concentrations of cGMP. The levels of NOx in AF rose progressively throughout the third trimester.
Mechanisms for increased blood flow in the fetoplacental circulation during this critical period of fetal development involve both vasodilation and neovascularization within the placenta [1–5]. A physiological cause-and-effect role for NO in placental vascular function and placental blood flow has been demonstrated by studies in which NOS inhibition potentiated vasoconstriction of human stem villous placental arteries and reduces ovine placental blood flow by increasing umbilical vascular resistance [13, 14]. Although eNOS expression has been demonstrated in human umbilical and placental vessels [7, 9, 17, 19–21] and in an ovine placental artery endothelial cell line [16], the ontogeny of the quantitative regulation of its physiological in vivo expression during the third trimester has not been previously described.
During the third trimester, fetal weight increases rapidly, accelerating as gestation advances until near term, when the rate of fetal weight gain slows considerably. The current data suggest an important role for local endothelium-derived NO in the vasodilation of placental vasculature during this critical period of fetal growth and development. Levels of eNOS expression during the early third trimester were unaltered between 110 and 120 days but exhibited a dramatic rise by 130 days, a time of maximal rate of individual fetal growth velocity (kg/day) in these animals. We observed a decline in eNOS expression at 142 days of gestation, which is very near term, when the rate of fetal growth slows substantially. In addition, concentrations of AF cGMP, the physiologic mediator of NO action in VSM [9–12], exhibited the same developmental pattern as eNOS (r2 = 0.78), peaking significantly at 130 days of gestation in AF and then falling at term gestation. Because there are very dynamic physiological changes that occur towards term, longitudinal daily samples of AF for cGMP levels would have revealed when the fall in AF cGMP actually occurred. However, even with this limitation of the current study design in which we could not obtain longitudinal daily data for fetal growth, eNOS levels, and AF cGMP, our findings suggest that the increases in eNOS expression from 120 to 130 days are accompanied by an increase in the production of NO locally within the fetoplacental vasculature, resulting in cGMP-mediated relaxation of VSM tone and thus placental vasodilation.
In the present study we hypothesized that the AF levels of nitrates and nitrites (NOx), studied as an index of NO, would parallel the expression of fetoplacental artery eNOS and AF cGMP levels. Although, we did detect progressive increases in nitrates and nitrites in AF throughout the third trimester and the levels of AF NOx and cGMP were moderately correlated (r2 = 0.27), their patterns of change were not identical. The NO levels in the AF may reflect both vascular and placental NO production secondary to a rise in inducible NOS (iNOS) expression; in recent studies we have observed significant iNOS protein levels in the cotyledon [8]. Also, renal excretion of nitrates and nitrites into the fetal urine may have obscured subtle changes in fetoplacental vascular/cotylendonary NO, or the NOx in the AF may reflect fetal renal vascular NO production more so than fetoplacental vascular or placental NO. This latter hypothesis is supported by our recent observations that, as seen for endothelial eNOS levels in the present study, fetoplacental cotyledonary NO production in tissue culture increases during the third trimester and peaks at 130 days of gestation [8]. It is more likely that the cGMP in AF may not be reflective of only rises in vascular/placental NO production with changes in eNOS expression. Levels of AF cGMP could also reflect increased sensitivity or expression of soluble guanylate cyclase, the enzyme stimulated by NO to increase cGMP production [12]. Production of cGMP in ovine uterine arteries [12] and fetoplacental artery endothelial cells [24] can derive from the stimulation of particulate guanylate cyclases via natriuretic peptide exposure. Additional studies will be needed to determine whether fetoplacental artery NOS-specific activity is indeed increased during the third trimester and whether soluble and/or particulate guanylate cyclase sensitivity is also altered to account for the AF rise in cGMP in the current study.
The current data are consistent with the findings in animal models showing that chronic inhibition of NO production during pregnancy induces fetal growth restriction [25] via decreases in fetoplacental blood flow [14], demonstrating that NO is vital to the maintenance of vasodilation in the fetoplacental circulation. Our observations also have important clinical relevance regarding fetal growth disorders such as intrauterine growth retardation, which is associated with reduced fetoplacental blood flow and has been partly attributed to decreases in placental NO production [26, 27]. The present data suggest that NO-mediated vasodilation is developmentally regulated during the third trimester and that this change appears to be especially important between 120 and 130 days of ovine gestation (82%–90% of term), when placental artery eNOS expression and AF cGMP levels are elevated significantly.
The mechanism(s) by which the increase in fetoplacental artery eNOS protein expression is associated with concurrent increases in AF NOx and AF cGMP remains to be determined. Angiogenic growth factors of placental origin probably directly increase the production of NO [1–5]. The level of the angiogenic growth factor bFGF is increased in ovine placenta and its vasculature [5] in a fashion that parallels the current increase in eNOS expression and AF cGMP (Fig. 4). Direct comparisons can readily be made between these experiments because the same animals were used in both studies. Moreover, a cause-and-effect relationship is implied by our recent observations that bFGF dose dependently increases eNOS protein and mRNA expression [28] and NO production (Zheng et al., unpublished communication) in cultured ovine fetoplacental artery endothelial cells. An additional mechanism may be involved in parallel with the rise in AF cGMP; bFGF treatment increases the atrial natriuretic peptide (ANP) sensitivity of ovine fetoplacental artery endothelial cells to increase cGMP upon stimulation of the ANP-A particulate guanylate cyclase-A receptor [24].
ACKNOWLEDGMENTS
We thank Jing Zheng and Lisa Modrick for their help with the immunohistochemistry. We also acknowledge Terrance M. Phernetton for his assistance with the collection of the tissues, Cindy Goss for helping in the preparation of this manuscript, and Terry Jobsis, Lee Sherven, and Erica Ohst for their expert assistance with animal care.
FOOTNOTES
First decision: 27 September 2000.
1 This investigation was supported by National Institutes of Health grants HL49210, HL57653, HD33255, HL56702, and HD38843 and American Heart Association-Wisconsin Affiliate 95GS-74. Dr. Sheppard was a fellow in Maternal Fetal Medicine. 
2 Correspondence: Ronald R. Magness, Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Perinatal Research Laboratories, 7E Meriter Hospital/Park, 202 S. Park St., Madison, WI 53715. FAX: 608 257 1304; rmagness{at}facstaff.wisc.edu
3 Current address: Scott and White Ob/Gyn Clinic, Temple, TX 76508.
Accepted: December 28, 2000.
Received: August 8, 2000.
REFERENCES
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|
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