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Endocrinology Vol. 138, No. 1 490-498
Copyright © 1997 by The Endocrine Society

ARTICLES

Pregnancy Induces an Increase in Angiotensin II Type-1 Receptor Expression in Uterine But Not Systemic Artery Endothelium1

Ian M. Bird, Jing Zheng, Jacqueline M. Cale and Ronald R. Magness

Departments of Obstetrics and Gynecology, Perinatal Research Laboratories (I.M.B., J.Z., J.M.C., R.R.M.), and Meat and Animal Science (R.R.M.), University of Wisconsin - Madison, Madison, Wisconsin 53715

Address all correspondence and requests for reprints to: Ian M. Bird, Ph.D., University Wisconsin - Madison, Department Obstetrics and Gynecology, Perinatal Research Laboratories, 7E Meriter Hospital/ Park, 202 South Park St., Madison, Wisconsin 53715. E-mail: IMBird{at}FACSTAFF.Wisc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During pregnancy, the uterine artery demonstrates refractoriness to vasoconstriction by infused angiotensin II (AII). AII increases prostacyclin (PGI2) production by uterine artery endothelium from pregnant ewes, and this response is mediated via the AT1 receptor (AT1-R). This response is also unique to pregnancy because AII does not stimulate PGI2 production by uterine artery endothelium from nonpregnant ewes. We therefore hypothesize that the increase in uterine artery PGI2 production in response to AII in pregnancy is associated in part with a concomitant increase in AT1-R expression in uterine artery endothelium. Endothelium-derived protein was directly removed from the lumenal surface of freshly isolated uterine and systemic (omental) arteries from nonpregnant and pregnant ewes. AT1-R expression was then measured in both the endothelium-derived fraction and endothelium-denuded vascular smooth muscle (VSM) fraction by Western analysis. AT1-R was detected as 54- and 65-kDa proteins in all samples, as well as adrenal cortex control. AT1-R expression increased more than 8-fold in uterine artery endothelium of pregnant ewes over that in nonpregnant ewes at each of four gestational ages (P < 0.05 at 110, 120, 130, 142 days, n = 4 each vs. n = 6 nonpregnant). No significant differences were seen, however, from 110 to 142 days of gestation. In contrast, whereas the level of AT1-R staining in omental artery endothelium in nonpregnant ewes was higher than in uterine artery, AT1-R increased less in pregnant ewes (2-fold) and only reached significance over nonpregnant values at 110 and 120 days, or when data was combined irrespective of gestational age (P < 0.05). Although AT1-R was also detected in uterine and omental artery VSM, little or no change in expression was observed in pregnancy. Results were confirmed by immunohistochemical staining of arterial cross sections, and the increase in AT1-R expression in uterine artery endothelium was confirmed by RT/PCR amplification of AT1-R messenger RNA from collagenase dispersed cells (n = 4 pregnant vs. n = 4 nonpregnant, mean 20-fold increase, P < 0.028). We conclude that increased uterine artery endothelial PGI2 responsiveness to AII during pregnancy is indeed associated with a correspondingly marked and localized increase in expression of the endothelial AT1-R receptor. We believe our findings allow a more detailed understanding of the molecular mechanisms that underlie increased uterine blood flow that is so central to the normal development of the growing fetus, and on dysfunction may lead to conditions such as preeclampsia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREGNANCY IS A time of marked changes to the maternal cardiovascular system, resulting in a 20- to 30-fold increase in uteroplacental blood flow and so a dramatically increased supply of oxygen and nutrients for the growing fetus. In both pregnant sheep and pregnant women, there is a reduction in the systemic pressor effect of infused angiotensin II (AII), even though circulating levels of AII are elevated 4- to 5-fold in pregnancy (1). The uterine vasculature, however, is even more refractory to AII-induced vasoconstriction than the systemic circulation (2, 3, 4, 5). The net result of these changes, together with remodeling of the uterine artery to increase its physical size (6, 7), is a marked reduction in uteroplacental vascular resistance and a dramatic increase in uteroplacental blood flow.

AII has long been regarded as a potent vasoconstricting agent intimately involved in the control of vascular tone. In the aorta and many systemic arteries in adult nonpregnant animals, the VSM expresses the AII type-1 receptor (AT1-R) (8, 9, 10), a subtype that is known from expression studies to couple via G proteins to activation of phosphoinositidase C and elevation of intracellular Ca2+ ([Ca2+]i) (11), so causing contraction. In addition, vascular contraction can be attenuated by secretion of vasorelaxing factors such as PGI2 and NO from the endothelial cells lining the inner lumenal surface. It is now increasingly apparent that the secretion of these endothelium-derived vasorelaxing factors is often under the direct control of many hormones classically associated with VSM contraction, including AII, and that pregnancy in particular appears to selectively augment this endothelial response (reviewed in Refs. 12 and 13). In both pregnant sheep and pregnant women, the uterine artery demonstrates both increased PGI2 production and attenuated increases in uterine vascular resistance in response to infused AII, both of which can be reversed by the cyclooxygenase inhibitor, indomethacin (5, 14, 15). These responses appear unique to pregnancy because PGI2 production is not observed in the uterine artery in the nonpregnant state (16, 17) and is unique to the uterine artery because AII does not increase PGI2 production from omental (systemic) artery segments from either nonpregnant or pregnant ewes (16, 18). Moreover, the endothelium is the sole source of this AII-induced increase in uterine artery PGI2 production because removal of the endothelium abolishes this response (17, 18). Of particular interest, however, is the additional finding that this normal pregnancy-induced increase in PGI2 production (19) and reduced pressor response to AII is also impaired in preeclamptic women (20). These observations strongly imply a key role for this normal adaptive response in maintaining maternal health as well as optimal intrauterine environment.

From recent studies of the effects of AII receptor subtype-selective antagonists on PGI2 production by uterine artery segments, it is now clear that the AII-induced increase in uterine artery endothelial PGI2 production is mediated via the AT1-R receptor. Furthermore, although AII stimulated this PGI2 response in uterine artery segments only from pregnant, but not nonpregnant ewes, an increase in production of PGI2 in response to exogenous arachidonate or A23187 (a Ca2+ ionophore) was observed in both groups (17). The implication of these findings, therefore, is that the pregnancy specific increase in PGI2 production by uterine arteries in response to AII may relate to either an increased coupling of existing endothelial AT1-R to second messenger signaling pathways, or an increase in endothelial AT1-R expression. In the current study, we have directly evaluated the latter hypothesis, and report for the first time that pregnancy is indeed associated with increased endothelial AT1-R expression at the level of both cell protein and messenger RNA (mRNA) in uterine artery which far exceeds that observed in omental (systemic) artery.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental design
Uterine and systemic (omental) arteries were obtained from Polypay and mixed western breed nonpregnant sheep (n = 6) and pregnant ewes at 110, 120, 130, and 142 days of gestation (n = 4 each). Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison Research Animal Care Committees of both the Medical School and the College of Agriculture and Life Sciences and follow the recommended AVMA guidelines for humane treatment and euthanasia of laboratory farm animals. Ewes were subjected to nonsurvival surgery using iv general anesthesia (Na+Pentobarbital; Nembutal; 25–50 mg/kg) that was titrated to maintain tissue perfusion and oxygenation during the time of tissue collection. During nonsurvival surgery/euthanasia, uterine and omental arteries were rapidly (10–15 min) obtained for study. Briefly, the fetuses were excised via hysterotomy, the umbilical cord clamped and the fetuses weighed and crown-rump length determined. The uterus with the entire broad ligament, mesometrium, mesovarium, and mesosalpinx, and the greater and lesser omentum were then rapidly excised and placed in ice-cold PBS (10 mM PBS; 8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, pH 7.4). Uterine and omental arteries were dissected free of connective tissue, fat and veins and the arteries were thoroughly rinsed free of blood.

Immunohistochemistry
At the time of euthanasia, intact uterine and omental artery segments and slices of adrenal were placed in 4% formaldehyde in sodium cacodylate buffer (0.1 M, pH 7.4), and fixed overnight. Tissues were embedded in paraffin, cut into sections (6 µm), and mounted on poly-lysine coated glass slides. Following deparafinization and graded rehydration, sections were incubated in 3% H2O2 in methanol for 15 min to quench endogenous peroxidase activity. AT1-R antibody (rabbit polyclonal IgG fraction (2 µg/ml), Santa Cruz Biotechnology, Santa Cruz, CA) was then applied. Staining was detected using a biotinylated secondary antibody in combination with the avidin-biotin-peroxidase (ABC) method (ELITE ABC kit, Vector Laboratories, Burlingame, CA) with 3,3'-diaminobenzidine (DAB) as chromagen (21). As controls, adjacent sections were alternatively treated with a control rabbit IgG fraction (2 µg/ml, Vector), without primary antisera, or without second antibody. Control sections were also prepared from the ovine adrenal, which express AT1-R in the cortex (positive control) but not medulla (negative control). These control sections were routinely stained in parallel with each batch of uterine and omental artery slides prepared from both nonpregnant and pregnant animals. On parallel arterial sections we also performed immunohistochemistry for ecNOS as a specific endothelial marker, so allowing us to confirm the cellular location of endothelial AT1-R staining (not shown; see Ref.22).

Western analysis
We have previously described and validated a rapid isolation procedure to obtain endothelium devoid of VSM contamination (22). Intact artery segments were opened longitudinally, and the lumenal endothelium/tunica intima was mechanically removed as previously described (22) and 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 pheylmethylsulfonylfluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin]. The lumen of the remaining vessel was then rubbed with a wet cotton swab to remove any remaining endothelium; this endothelium-denuded vessel represents primarily VSM (17). Endothelium-derived proteins and denuded arteries were immediately snap frozen in liquid nitrogen. Endothelial and denuded artery preparations were subsequently solubilized in lysis buffer by homogenization and sonication. Solubilized protein was quantified using a modified Lowry assay procedure, (Bio-Rad). Proteins (50 µg/lane) were then separated by size on 7.5% polyacrylamide gels (100 V, 2.5 h, Mini Protean II, Bio Rad) alongside positive controls (adrenal cortex homogenate, 10 µg/lane) and rainbow mol wt markers (Bio Rad) before transfer to Immobilon P membrane (100 V, 2 h). The Immobilon P membrane was then probed for AT1-R using the Enhanced Chemiluminescence (ECL) reagent detection system, as described by Amersham, and exposed to hyper-film (5 min). The AT1-R specific antisera (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a dilution of 1:750, and second antibody (donkey antirabbit/HRP conjugate; Amersham) was used at 1:5000 dilution. Levels of AT1-R were then quantified by scanning densitometry (Bio-Rad 670 scanning densitometer) and expressed relative to adrenal standard (arbitrarily set at 10 U).

Isolation of uterine artery endothelial cells and extraction of total cellular RNA
Endothelial cells were first isolated by collagenase digestion from four nonpregnant and four pregnant ewes, and RNA immediately extracted (below). Uterine arteries obtained at the time of euthanasia were flushed free of blood using M199 medium, before tying off arterial branches, clamping off the larger diameter end, and inflating with M199 containing 5 mg/ml collagenase B (Boehringer Mannheim) and 0.5% BSA through a luerlock three-way tap. Digestion was allowed to proceed at 37 C for 55 min before flushing the collagenase solution and endothelial cell sheets from the inner surface of the vessel. We have previously shown that cells isolated in this way are endothelial in origin and of high viability and purity through their uniform expression of endothelial nitric oxide synthase [ecNOS, an endothelial marker in the uterine artery; (22)], their cobblestone morphology in primary culture, and by uptake of acetylated LDL as visualized under UV excitation (23). For extraction of total cellular RNA, freshly isolated endothelial cells were washed in fresh M199 and pelleted by centrifugation before solubilizing in 1 ml RNAzolB (Cinna Biotecx, Houston, TX). After addition of 150 µl chloroform, and phase separation by centrifugation (12,000g, 20 min) the upper aqueous phase was removed, extracted twice with phenol/chloroform/isoamyl alcohol using Heavy grade phase lock gel (5-Prime, 3-Prime, Boulder CO) and finally mixed with 110% by volume of isopropanol. RNA was then precipitated by standing at -20 C for 1 h before recovery by centrifugation (12,000g, 30 min), and washing of the pellet in 75% Ethanol. RNA was then solubilized in molecular biology grade water (5-Prime, 3-Prime, Bolder CO) and quantified by spectrophotometry.

RT/PCR AT1-R mRNA mass assay
AT1-R mRNA was quantified by coupled RT/PCR amplification in a single tube assay, based on the method of Mallet et al. (24). All reagents were obtained from GIBCO-BRL (Gaithersburg, MD). Total cellular RNA (1 µg per tube) was incubated in a 50 µl final volume containing 1 x PCR buffer, 3 mM MgCl2, 10 nmoles each dATP, dTTP, dCTP, and dGTP, and 30 pmol each of forward and reverse temperature matched primers, targeting amplification from the 5' terminus of the ovine AT1-R untranslated region into the protein coding region. The final product was 718 bases and, by homology to the human and bovine sequence, spanned at least two intron sites; thus genomic contamination would not result in a false signal because it would be of considerably greater size. Amplification was performed in the presence of 1 µl AMV Reverse Transcriptase (2.5 U) and 1 µl of Taq Polymerase (5 U), except for RT- controls, which only contained Taq Polymerase. The cycle program used was anneal 65 C, 10 min, reverse transcription 50 C, 10 min, denature 94 C, 2 min, followed by 35 cycles of amplification using 94 C, 1 min; 66 C, 30s; 72 C, 1 min. Final products were extended to full length by incubation at 72 C for 10 min. Controls for each assay included ovine adrenal cortex RNA (RT+ and RT-) and a standard curve containing known copy numbers of AT1-R cDNA target sequence (Note that the presence or absence of mRNA species other than the reverse primer target has little effect on the standard curve and so was not necessary for the standards - personal communication, M. Wiltbank). At the end of the assay 10 µl of products were separated on a 1% TAE gel and transferred to MagnaGraph hybridization membrane (Molecular Separations Incorporated) for Southern blotting against a probe encoding the bovine AT1-R protein coding sequence, generated by asymmetric PCR as described previously (25). After hybridization, membranes were washed once in 2 x SSC/0.1% SDS for 30 min and twice in 0.1X SSC/0.1% SDS (2 x 30 min) before drying and direct exposure to a phosphorimager (Bio-Rad BI screen, 1 h) for direct quantification (Molecular Analyst v1.4, Bio-Rad). Data were calculated as copy number AT1-R mRNA per µg total cellular RNA from the standard curve (see Fig. 5Go).



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  		Figure 5. Validation of coupled RT/PCR assay. Left, The effects of cycle number on AT1-R RT/PCR product are shown using RNA from adrenal cortex (total RNA). Note the uniform increase in signal with cycle number. Right,Standard curve generated for the assay using known copy numbers of AT1-R cDNA template. Note also the threshold of detection at 100 copies and the upper limit at 1010 copies. Regression analysis revealed an r2 value of 0.989, with P < 0.0001. Inset right, Relative levels of AT1-R mRNA in total RNA from liver, kidney, and adrenal (1 µg each) are shown. AT1-R transcripts/µg total RNA were 0.998 x 106, 2.8 x 106, and 82.5 x 106, respectively. The result of PCR amplification of adrenal RNA in the absence of reverse transcriptase (RT-) is also shown (negative control), giving a result of 1364 transcripts/µg (baseline; 0.0017% RT+ value).

 
Statistical analysis
Data were analyzed by one- and two-way ANOVA or Student’s t test, as appropriate. Means were compared by Student Newman-Keuls multiple range test. Data presented are the means ± one SE. Results were considered significant at the P < 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have characterized an antisera raised against a conserved peptide sequence in the AT1-R (Santa Cruz Biotechnology, Santa Cruz, CA) that cross-reacts with the sheep receptor protein. We have validated this antisera by immunohistochemistry of the ovine adrenal gland that is known to express AT1-R in the zona fasciculata/reticularis as well as the zona glomerulosa (26, 27, 28). Adrenal sections were stained with the AT1-R antisera (Fig. 1Go) or with an equivalent nonimmune IgG control serum (not shown). Our findings demonstrate the specificity of the AT1-R antisera to the subcapsular zona glomerulosa cells (ZG) together with a reduced level of staining through the zona fasciculata and reticularis (ZF and ZR, respectively). No immunostaining was observed in the outer capsule or adrenal medulary cells (M), consistent with the previously reported lack of AII-binding in adrenal medula of the human, monkey and cow (29, 30).



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  		Figure 1. Upper panel, Immunohistochemical staining of AT1-R in ovine adrenal cross sections: validation of antisera. Ovine adrenal tissue sections were prepared and stained with rabbit polyclonal AT1-R-specific antisera (IgG fraction) as described in Materials and Methods. Specific binding was detected using a biotinylated secondary antibody in combination with the avidin-biotin-HRP reagents (Vector Laboratories, Burlingame, CA) and diaminobenzidine stain. Positive staining is brown. Control sections stained either with a control IgG fraction, without primary antisera, or without second antibody, gave negative results (not shown). Note: C, Capsule; ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis; M, medulla. Lower panel, Immunohistochemical staining of AT1-R in uterine and omental artery cross-sections from nonpregnant and pregnant ewes: uterine artery and omental artery tissue sections were fixed and stained for AT1-R expression as described above. Positive staining is brown. Control sections of all vessels were stained in parallel either with a control IgG fraction, without primary antisera, or without second antibody; all gave negative results (representative uterine artery control IgG sections shown lower left).

 
In addition to immunohistochemistry, we have further validated this AT1-R antisera by Western analysis of tissue homogenates from liver, kidney, and adrenal cortex, all of which are classic target tissues known to express AT1-R but little or no AT2-R. Adrenal medulla homogenate was also used as negative control, which has been shown to express little or no detectable AT1-R (31, 32). Western analysis of 10 µg protein from each tissue (Fig. 2Go) reveals staining of one major band at 54 kDa, with a lighter band visible at 65 kDa in liver, kidney, and adrenal cortex homogenates; in contrast, these bands were correspondingly very faint or absent in the lane containing homogenate of adrenal medulla. The relative order of magnitude of AT1-R expression was found to be adrenal medulla<<<liver<kidney<adrenal cortex. In addition, to determine whether this technique combined with transmission scanning densitometry could give accurate quantification of AT1-R protein, a Western blot of serially diluted adrenal cortex homogenate was prepared and analyzed as above. Linearity for accurate quantification was confirmed for up to 10 µg of adrenal cortex homogenate protein, with a lower limit of sensitivity of 1 µg adrenal cortex homogenate protein at 1 min exposure time and 0.25 µg at 5 min. In this range (0–10 µg), first order regression analysis by the least squares method gave a high degree of correlation (1 min, r2=0.989 or 5 min r2=0.941, both P < 0.0001).



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  		Figure 2. Western analysis of AT1-R protein expression in ovine liver, kidney, adrenal cortex and adrenal medulla. Homogenates of ovine liver, kidney, adrenal cortex, and adrenal medulla were separated by electrophoresis (10 µg/lane, 7.5% SDS PAGE gel). After transfer to Immobilon P membrane immunoblotting was performed using AT1-R antisera with ECL detection, as described. Results are shown alongside a mol wt scale derived from Rainbow mol wt markers run on the same gel. The range of sensitivity of this technique is also indicated by the lower panel showing the results of analysis of serially diluted adrenal cortex homogenate. The lower limit of detection was at 0.25 µg adrenal cortex homogenate at 5 min exposure, with linearity over the range 0–10 µg (r2 = 0.941, P < 0.0001).

 
Uterine artery cross-sections from nonpregnant and pregnant ewes of 110–142 days of gestational age (term = 145 ± 3 days) were immunohistochemically stained for AT1-R as shown above for adrenal sections. In addition, cell nuclei were counterstained with hemotoxylin, showing blue. The results of staining uterine artery cross sections from a representative nonpregnant ewe, as well as a 130-day pregnant ewe are shown in Fig. 1Go. Positive AT1-R staining was apparent on the endothelial cells in nonpregnant controls, with some faint staining in the underlying VSM. However, in uterine arteries from pregnant ewes, AT1-R staining was much stronger at the level of endothelium, with some additional diffuse staining throughout the VSM. In both cross-sections, however, the intervening lamina propria remained unstained. Whereas space does not permit further photographs, similar results were observed in other nonpregnant ewes of both follicular and luteal phases, and in other pregnant animals from 110, 120, 130, and 142 days of gestation; all IgG controls run in parallel were negative (see lower left panels, Fig. 1Go). Parallel studies performed on omental arteries (lower right panels, Fig. 1Go) indicate a uniformly higher level of expression of AT1-R in both endothelium and VSM in nonpregnant ewes compared with uterine arteries, but a much less marked or no increase in expression was observed in omental artery endothelium of pregnant animals compared with nonpregnant controls.

In the light of our findings from immunohistochemical studies concerning pregnancy-induced uterine artery AT1-R expression, we chose to further analyze these changes by using the recently developed direct endothelial isolation technique, together with Western analysis, so allowing quantification of the 54-kDa band by scanning transmission densitometry. Representative data from Western blots are shown in Fig. 3Go, and the means of combined data from all animals studied are shown for uterine artery and omental artery endothelium and VSM in Fig. 4Go. The most striking result is that pregnancy increased uterine artery endothelial AT1-R expression more than 8-fold over that in nonpregnant animals, consistent with the results of immunohistochemical analysis. This increase was significant at each of the four gestational ages studied (P < 0.01, n = 6 nonpregnant, n = 4 each gestational age), but no significant differences were seen with increasing gestation from 110 to 142 days. Combined data from pregnant ewes, irrespective of gestational age, also showed significance over nonpregnant values (P < 0.0001). In contrast, whereas a smaller but significant increase in AT1-R expression was detected at 110 and 120 days of gestation in omental artery endothelium, this was not maintained throughout the third trimester of pregnancy, and when data was combined regardless of gestational age, only a 2-fold increase was observed relative to nonpregnant controls (P < 0.05). Furthermore, whereas the initial level of AT1-R staining in omental artery endothelium in nonpregnant animals was initially higher than that in uterine artery endothelium, (Fig. 4Go, P < 0.05, denoted "a"), the level of AT1-R expression in omental artery endothelium from pregnant ewes was significantly less than that in the uterine artery endothelium from pregnant ewes (Fig. 4Go, P < 0.05, denoted "b"). Similar relative changes were also observed for the 65-kDa band (data not shown), supporting its identity as an alternate form of the AT1-R.



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  		Figure 3. Western analysis of AT1-R protein expression in uterine and omental artery endothelium and VSM isolated from nonpregnant or pregnant ewes. Endothelium (Endo) or vascular smooth muscle homogenates (VSM) obtained from the uterine artery (UA) or omental artery (OA) of nonpregnant (NP, n = 6) and pregnant (110, 120, 130, and 142 days of gestation, n = 4 at each age) ewes were separated by electrophoresis (50 µg/lane, 7.5% SDS PAGE gel). After transfer to Immobilon P membrane immunoblotting was performed using AT1-R antisera and ECL detection. Representative blots of each preparation are shown. The results from a positive control (10 µg adrenal cortex) are also shown (Adr, right lane).

 


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  		Figure 4. Quantification of changes in the expression of AT1-R protein in uterine and omental artery endothelium and VSM isolated from nonpregnant and pregnant ewes. Results from multiple blots illustrated in Fig. 3Go were quantified (54-kDa band) by transmission densitometry and normalized to the intensity of the adrenal control run on the same gel. Combined data are shown (arbitrary units) together with statistical analysis performed on the combined data (ANOVA, P < 0.05 or as shown vs. nonpregnant controls. "a" denotes significant difference relative to uterine artery endothelium in nonpregnant ewes; "b" denotes significant difference relative to data for uterine artery endothelium from all pregnant ewes, irrespective of gestational age).

 
While Western analysis suggested AT1-R expression was also increased in the uterine artery VSM, the magnitude of the response was considerably less than that observed in the uterine artery endothelium and did not show a consistent increase across the third trimester. Combined data from all pregnant ewes showed only a 2.5-fold increase in uterine artery VSM AT1-R expression relative to nonpregnant controls (P < 0.01). There were no consistent changes in AT1-R expression in omental artery VSM through the third trimester, nor a significant change when all data from pregnant ewes were combined. Thus, the uterine artery endothelium showed the lowest level of AT1-R expression in the nonpregnant ewe, but increased more than 8-fold during the third trimester to the highest level of expression, which was significantly greater (P < 0.05, denoted "b") than that seen in omental artery endothelium or in both uterine and omental artery VSM.

Previous studies have suggested that changes in AT1-R expression are a reflection of, and preceded by, changes in the corresponding mRNA levels (25). For this reason, we isolated uterine artery endothelial cell RNA and determined AT1-R mRNA levels by quantitative coupled RT/PCR. The assay was optimized to show uniform cycle dependent increases in signal for up to 35 cycles with adrenal cortex total RNA as template (Fig. 5Go). In addition, control RNA samples isolated from liver, kidney, and adrenal cortex gave the same relative order of magnitude of AT1-R mRNA (Fig. 5Go, inset) as is observed for receptor protein (Fig. 2Go), and no signal was observed from adrenal RT- control. Coupled RT-PCR analysis of AT1-R mRNA levels in uterine artery endothelium confirmed that the levels in endothelium from pregnant ewes were consistently increased (mean 19.3-fold, P < 0.028) over that detected in nonpregnant ewes (Fig. 6Go), in agreement with the observed elevation of AT1-R protein observed above.



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  		Figure 6. Quantification of uterine artery endothelial AT1-R mRNA levels in pregnant and nonpregnant ewes. AT1-R mRNA levels were quantified by RT-PCR amplification of RNA isolated from uterine artery endothelial cells of nonpregnant (n = 4) or pregnant (n = 4) ewes, exactly as described. Samples were run alongside the liver, kidney and adrenal controls and the standard curve shown in Fig 5Go. Results are shown as mean ± SE of the data; *, P < 0.05 pregnant > nonpregnant controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During pregnancy, there are marked changes in the cardiovascular system that largely serve to redistribute blood flow to the uteroplacental unit, and so provide an ever increasing supply of oxygen and nutrients to the developing fetus while at the same time maintaining blood pressure within acceptable limits for the mother. The increased perfusion of the uteroplacental unit continues to increase exponentially during the later portion of gestation, in spite of a 3- to 4-fold increase in circulating levels of the normally potent vasoconstrictive agent, AII, which is considered an important factor in the maintenance of vascular tone (1, 12, 33, 34). Concomitant with this, the peripheral vasculature exhibits increased refractoriness to the pressor effect of infused AII (20, 35). Furthermore, the effect of infused AII on the uterine vasculature is even less pronounced than on the systemic vasculature (2, 3, 4, 5). One previously proposed mechanism for such changes is a reduced expression or internalization of VSM receptors to AII. Binding studies have demonstrated, however, that AII receptor density in VSM membrane preparations is not changed by pregnancy in either the mesenteric or uterine artery (33). Further characterization of AII receptor subtypes in VSM membrane preparations of uterine, mesenteric, renal, mammary arteries, and aorta from nonpregnant and pregnant sheep using AII receptor subtype-specific antagonists have also shown that while uterine artery VSM expresses a surprising predominance of AT2-R and all other arteries studied express predominantly AT1-R, pregnancy has little effect on their expression level in any vessel (10, 33, 36, 37). Whereas these studies focused on possible changes in VSM receptor expression, and as such were performed on membranes preparations from arteries first denuded of endothelium, binding data were not presented for the endothelium itself. An alternative mechanism for uterine vascular refractoriness to the pressor effects of AII during pregnancy, follows from the known role of AII in stimulating an increase in endothelium-derived vasodilators such as prostacyclin (PGI2) and nitric oxide (NO) (12, 34, 38). This PGI2 response in particular appears vessel-specific because AII stimulates an increase in PGI2 production by the uterine artery but not omental artery of pregnant sheep, and pregnancy-specific because neither omental nor uterine artery from nonpregnant ewes produce PGI2 in response to AII (16, 17, 18). This PGI2-production by uterine artery in pregnancy also occurs in an endothelium-dependent manner, (17, 18), and more detailed examination has shown that this endothelial PGI2 response by uterine artery is specifically mediated through AT1-R and not AT2-R (10). Furthermore, the failure of uterine artery endothelium in the nonpregnant state to produce PGI2 in response to AII is not due to a lack of endogenous substrate or cyclooxygenase because receptor-independent stimulation with Ca2+ ionophore (A23187) induces maximal PGI2 production, as does addition of endogenous arachidonate (17). This response, therefore, must be acquired during pregnancy through changes that are proximal to the cyclooxygenase pathway and thus must occur at the level of the AT1-R/signalling complex. It is possible that such a change may involve a change in expression of the AT1-R itself, or altered coupling of the receptor to the second messenger signaling system. We chose to directly investigate the hypothesis that the increase in uterine artery vasodilator production in response to AII in pregnancy is associated with a marked increase in AT1-R expression in uterine artery endothelial cells, both at the level of protein and mRNA. The recent availability of a polyclonal antisera to the AT1-R, which we have also validated in this study, has made possible for the first time the direct visualization of AT1-R expression in the uterine and systemic artery segments by immunohistochemistry, together with quantification of endothelial vs. VSM AT1-R expression at the level of western analysis. In addition the recent isolation and sequencing of the 5'-untranslated region of the ovine AT1-R cDNA by 5'-RACE (39) has made possible the quantification of pregnancy-induced changes of uterine artery endothelial AT1-R mRNA levels from small quantities of endothelium.

We have demonstrated the antisera specificity through immunohistochemical staining of ovine adrenal cross sections. The adrenal is a clearly zonated gland which has been extensively characterized and is a rich source of AT1-R in the cortex (highest in the zona glomerulosa, with lesser expression in the fasciculata and reticularis) but not the medulla. This has been determined in the human, monkey, rat, and cow by both immunohistochemical staining (32) and by [125I]-AII binding to tissue cross-sections (29, 30, 31). Our findings of high levels of staining in the outermost zona glomerulosa with lesser staining in the zona fasciculata and reticularis, but no staining of the adrenal medulla are in agreement with results obtained in parallel preparations using another AT1-R -specific monoclonal antisera [6313, courtesy G Vinson (40), data not shown] and are consistent with the results of Paxton et al. (32) and Chiu et al. (31), as well as the results of [125I]AII binding studies performed directly on bovine adrenal sections (29, 30). We have also further confirmed the antisera specificity by Western analysis, reporting AT1-R protein bands at 54 and 65 kDa. Previous studies have established that the AT1-R can be expressed with a variety of apparent mol mass by SDS-PAGE analysis, which appear tissue as well as species specific, and can be explained by monomer/dimer formation and/or glycosylation patterns (32). The apparent mol mass reported in our studies were very similar to those we reported previously in rat adrenal cortex using another AT1-R specific antisera (AT1-Blanka, 41) and also the mol mass reported by Paxton et al. (32) in studies of rat liver, kidney and adrenal, as well as rat aortic smooth muscle. We therefore conclude that the 54-kDa and 65-kDa bands reported here probably relate to alternate glycosylation with the predominant form being 54 kDa. The studies of Paxton et al. (32) also reported the same relative rank order of expression of AT1-R as we found here, i.e. liver<kidney<adrenal cortex, and we have further confirmed this relative order of expression at the level of AT1-R mRNA by RT/PCR mass assay. We take these positive results, combined with negative results against adrenal medulla, to be consistent with the antisera recognizing an AT1-R protein in ovine tissues.

Using this AT1-R specific antisera, we have also shown by immunohistochemistry that both uterine and systemic artery endothelium do indeed express AT1-R protein. However, quantification by Western analysis of changes in the 54-kDa band reveals that, while the level of endothelial AT1-R expression is significant and shows little alteration by pregnancy in the omental artery, the uterine artery endothelium shows comparatively lower levels of expression in the nonpregnant state and dramatically elevated levels (8- to 10-fold higher than nonpregnant) in the pregnant state, exceeding the level observed in the omental artery endothelium. In addition, the pregnancy-induced increase in uterine artery endothelial AT1-R expression is paralleled by an equally marked increase in AT1-R mRNA. To our knowledge, this is the first description of such a dramatically increased expression of any endothelial cell receptor being induced by a physiological state such as pregnancy. Thus the dramatic increase in AII-induced PGI2 production in the uterine artery that was previously reported to far exceed that in the systemic (omental artery) circulation during pregnancy is associated with a corresponding increase in endothelial AT1-R expression. In addition, although previous studies have revealed predominantly AT2-R expression in the uterine artery VSM (10, 36, 37), we confirm that AT1-R expression is still detectable in uterine artery VSM as well as in omental artery VSM but that the level is changed little by pregnancy in each case. Thus, the change in AT1-R expression in the endothelium far exceeds that in the VSM, consistent with the findings of in vitro studies of pregnancy-induced changes in uterine and omental artery PGI2 responses to AII in intact and denuded vessel preparations (17) and in the presence of subtype-specific antagonists (10). Moreover, the finding that AT1-R expression in VSM is greater in omental artery than in uterine artery is consistent with both animal and human studies that show infusion of AII increases systemic vascular resistance more than uterine vascular resistance (3, 4, 5).

With regard to gestational changes in AT1-R, the time-dated pregnancies used in this study spanned the third trimester, a time when dramatic increases in uterine blood flow are observed to meet the ever increasing demands of the growing fetus (34). Although we found pregnancy dramatically increased the level of uterine artery AT1-R expression over that in the nonpregnant state, we did not observe a change in elevated expression between 110–142 days gestation (term = 145 days). Thus, the increase in expression must have occurred before 110 days, and further studies will be necessary to determine exactly when this occurs.

Our finding that AT1-R expression in uterine artery endothelium is dramatically altered in pregnancy is, to our knowledge, an entirely novel observation. The mechanism of such a change remains unclear, but, as observed in other tissues (25, 28, 42), this change in receptor protein is accompanied by increased AT1-R mRNA. The question which remains, therefore, is by what regulatory mechanism does this increased level of AT1-R expression occur? Because pregnancy is associated with both increases in estrogen (43) and AII (1, 33, 44), both are possible candidates. Furthermore, AII increases AT1b-R expression in the rat adrenal (45), and estrogen increases AT1-R expression in the pituitary (46). However, the recent report that the AT1-R gene regulatory elements include a growth factor responsive consensus sequence (47) also raises the possibility for regulation by growth factors, and because this response is apparently unique to the uterine artery, such control is likely to be local rather than systemic. In support of this, we and others have recently observed that the ovine placentome secretes bFGF (48) as well as VEGF (49).

In conclusion, we have shown that pregnancy does indeed preferentially increase AT1-R expression on the uterine artery endothelium in a manner that exceeds that seen in the systemic vasculature. This increase in uterine artery endothelium AT1-R expression occurs at a time before 110 days of gestation, remains elevated throughout the third trimester, and is also accompanied by an increase in AT1-R mRNA level. These findings, together with the comparative lack of change in AT1-R expression on the uterine artery or omental artery VSM, may explain the increased level of local PGI2 production associated with normal pregnancy. In addition, we have recently reported that nitric oxide synthase expression is also elevated 2- to 4-fold in the uterine but not systemic vasculature during the third trimester of pregnancy (22). Together these findings clearly demonstrate that elevated uterine blood flow may be maintained throughout the third trimester through several changes at the level of endothelial cell function, an adaptive response that is so necessary to support the growing fetus and allow development to an optimum birthweight. Whereas our studies have primarily been performed on second and third generation distributing vessels, rather than on resistance vessels, previous autoradiographic studies of [125I]AII binding to intramyometrial resistance branches of the order of 100 µm diameter also show AT1-R sites on the cells lining the lumenal surface (10). These studies, however were not performed on vessels from pregnant sheep, and so the effect of pregnancy on AT1-R expression at this level still requires further investigation. Further studies will also be necessary to investigate whether the lack of uterine artery PGI2 production in response to AII in preeclamptic women is caused by a failure to increase uterine artery endothelial (50) AT1-R expression.


   Acknowledgments
 
The authors would like to express their thanks for expert technical assistance from T. M. Phernetton, C. E. Shaw, and D. S. Millican during the course of these studies. We also thank Professor Vinson for the use of the 6313 monoclonal AT1-R antisera.


   Footnotes
 
1 These studies were supported by grants from the American Heart Association (WI Affiliate 95-GB-41) and the NIH (HL-49210 and HD-33255). Back

Received June 24, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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