PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Hypertension. Author manuscript; available in PMC 2014 February 14.
Published in final edited form as:
PMCID: PMC3924857
NIHMSID: NIHMS430481

Estradiol-17β and its CYP450s- and COMT–Derived Metabolites Selectively Stimulate Production of Prostacyclin in Uterine Artery Endothelial Cells: Role of ER-α Versus ER-β

Abstract

Metabolism of estradiol-17β to 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol and 4-methoxyestradiol, contributes importantly to the vascular effects of estradiol-17β in several vascular beds. However, little is known about the role of estradiol-17β metabolites via the different estrogen receptors (ER-α/ER-β) on de novo endothelial prostacyclin and thromboxane production. We hypothesized that estradiol-17β and its metabolites, via ER-α and/or ER-β can enhance the prostacyclin/thromboxane ratio through the classic phospholipase A2, cyclooxygenase-1 and prostacyclin synthase pathway in ovine uterine artery endothelial cells (UAECs) in the pregnant (P-UAECs) versus the nonpregnant (NP-UAECs) state. Western analyses showed higher expression of phospholipase A2, cyclooxygenase-1 and prostacyclin synthase in P-UAECs whereas thromboxane synthase was higher in NP-UAECs. In P-UAECs, estradiol-17β, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol and 4-methoxyestradiolconcentration and time-dependently increased prostacyclin compared to control. Prostacyclin increase in NP-UAECs was of a lower magnitude. Estradiol-17β and its metabolites stimulated higher prostacyclin/thromboxane ratio in P-UAECs compared to NP-UAECs. Estradiol-17β-induced prostacyclin production was abrogated by the antagonists SC-560 (COX-1), U-51605 (Prostacyclin synthase), ICI 182 780 (ICI; both ER-α/β) and MPP (ER-α), but not by PHTPP (ER-β). Prostacyclin increases induced by its metabolites was abolished by SC-560 and U-51605 but unaltered by ICI, MPP or PHTPP. Our findings demonstrate that estrogen via primarily ER-α and its metabolites via ER-independent mechanisms influence the de novo endothelial biosynthesis of prostacyclin, which may be important in the regulation of vascular tone. These findings also shed light on the complexities of estrogen signaling via its metabolism and the functional heterogeneity of the estrogen receptors.

Keywords: prostacyclin, thromboxane, endothelium, estradiol metabolites, estrogen receptors

Introduction

Prostacyclin (PGI2) and thromboxane (TXA2), two major vasoactive prostanoids that exert opposing effects on vascular tone, are end products of the sequential reactions catalyzed by cytosolic phospholipase A2 (cPLA2), cyclooxygenase-1/2 (COX-1/2) and either PGI2 synthase (PGIS) or TXA2 synthase (TXAS) enzymes respectively.1 Classically, PGI2, a powerful vasodilator is produced mainly by vascular endothelial cells, whereas TXA2, a potent vasoconstrictor is principally produced by platelets.1 In this regard, little is known about whether endothelial cells can produce TXA2 and if this production plays a role in regulating the ratio of PGI2 and TXA2. PGI2/TXA2 ratio is considered of relevance in the regulation of physiologic and clinical vascular tone, with several studies showing that an imbalance in the generation of PGI2 relative to TXA2 is associated with hypertension, atherosclerosis and gestational vascular diseases such as preeclampsia.2-4

The dramatic rises in uterine blood flow during pregnancy are temporally associated with increases in de novo uterine vascular PGI2 secretion and are accompanied by augmented expression of uterine artery endothelial cPLA2, COX-1 and PGIS.5-8 These uterine blood flow rises are also partly mediated by increases in the plasma levels and actions of estrogen via the classical estrogen receptors (ERs).9-11 Infusion of estradiol-17β(E2β) in sheep causes rises in uterine blood flow, increases the uterine arterial expression of cPLA2, COX-1 and PGIS, which leads to the increase of the stable PGI2 metabolite 6-keto-PGF1α.7, 12, 13 In human umbilical vein endothelial cells (HUVECs), E2β has also been shown to selectively stimulate PGI2 production in vitro primarily via ER-α.14 However, less is known about the influence of E2β on endothelial TXA2 production and on the endothelial PGI2/TXA2 ratio.

The stimulatory effects of estrogen on uterine vascular endothelial PGI2 production maybe further modulated by its biologically active metabolites. E2β is sequentially metabolized by cytochrome P450s (CYP450s) to form the catecholestradiols, 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2), followed by metabolism of these catecholestradiols by catechol-O-methyltransferase (COMT) to form 2-methoxyestradiol (2-ME2) and 4-methoxyestradiol (4-ME2).15-18 In cultured HUVECs, 2-ME2 stimulates production of PGI2.19 Despite this knowledge, very little is known about the effects of E2β metabolites on endothelial PGI2 levels. Furthermore, nothing is known about the roles of the classical ERs on E2β metabolite-induced endothelial PGI2 levels. It is also unclear if E2β metabolites can alterTXA2 production and the PGI2/TXA2 ratio in endothelial cells and if this is altered in pregnancy.

Thus, we hypothesized that E2β and its metabolites 2-OHE2, 4-OHE2, 2-ME2 and 4-ME2 can augment the PGI2/TXA2 ratio via ER-α and/or ER-β, through the classical cPLA2-COX-1-PGIS pathway in uterine artery endothelial cells (UAECs) in the pregnant (P-UAECs) compared to the nonpregnant (NP-UAECs) state. Thus, we investigated:1) the expression of cPLA2, COX-1, PGIS and TXAS in P-UAECs versus NP-UAECs; 2) if treatments with E2β, 2-OHE2, 4-OHE2, 2-ME2 and 4ME2 can increase the PGI2/TXA2 ratio in pregnant uterine artery endothelial cells (P-UAECs) more than nonpregnant (NP)-UAECs; 3) the role of COX-1 and PGIS in P-UAEC PGI2 levels; and 4) the roles of ER-α and/or ER-β on PGI2 levels induced by E2β and its metabolites.

Methods

Cell Preparation and Culture

Animal use protocols and procedures were approved by the University of Wisconsin-Madison School of Medicine Research Animal Care Committee. Ovine NP-UAECs and P-UAECs were isolated, validated and cultured from nonpregnant (luteal n=2 and follicular n=2) and late gestation (120 -130 days; term, 147 days; n=4) ewes as previously described.6, 17, 18 At passage 4, ~70% confluence and serum starved, cells were lysed for Western blotting or transferred to 6-well plates for treatments as needed for respective experiments.

Protein Extraction and Western Immunoblotting

Protein extraction and Western Immunoblot analyses were performed as described previously.6, 17, 18 cPLA2, COX-1, PGIS and TXAS expressions were detected using mouse anti-cPLA2, rabbit anti-COX-1, rabbit anti-PGIS or rabbit anti-TXAS (1:1000) and respective secondary antibodies (1:2000). β-actin was used as loading control. Positive control was only utilized for the expression of TXAS using human platelet lysates. Human platelet lysates were a generous donation from the Platelet and Neutrophil Immunology Lab, Blood Center of Wisconsin.

Experimental Treatments, Blockade of Enzymes and Receptors

All experiments were performed in quadruplicates and replicated in at least four different P-UAEC (n=4) and NP-UAEC (n=4) preparations. For time and concentration-response studies, P-UAECs in 6-well plates were serum starved (24 hours) in EBM, washed with serum free EBM and the medium was replaced with EBM vehicle (Control) or EBM containing 0.1, 1, 10 or 100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, 4-ME2 or Ca2+ Ionophore (A23187; positive control) for 0, 2, 4, 8, 12 or 24 hrs. For nonpregnant versus pregnant concentration-response studies, NP-UAECs and P-UAECs in 6-well plates were serum starved (24 hours) in EBM, washed with serum free EBM and medium was replaced with EBM vehicle (Control) or EBM containing 0.1, 1, 10 or 100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, 4-ME2 or A23187 for 12 hrs based on time course studies. COX-1 or PGIS blockade was performed by pretreating P-UAECs with SC-560 and U-51605 (1μmol/L, 1hr), respectively followed by E2β, 2-OHE2, 4-OHE2, 2-ME2, 4-ME2 or A23187 treatments at optimal dose (determined from concentration-response curves). ERs were blocked by pretreating P-UAECs for1 hour with 1 μmol/L of the nonselective ER antagonist ICI 182,780, ER-α–selective antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP), or the ER-β–selective antagonist 4-[2-phenyl-5,7-bis (trifluoromethyl) pyrazolo [1,5-a]pyrimidin-3-yl]phenol (PHTPP) followed by treatments with EBM vehicle or EBM containing 0.1, 1, 10 or 100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, 4-ME2 or A23187 for optimal time determined from time courses.

PGI2 and TXA2 Assays

Following steroid treatments, media from individual 6-well plates were collected to measure production levels of PGI2 or TXA2 by using enzyme immunoassay kits (Cayman Chemical, Ann Arbor, MI). Indices of both PGI2 and TXA2 levels were calculated from measuring their stable non-enzymatic hydrolysis products, 6-keto-PGF1α and thromboxane B2 (TXB2) respectively in duplicates. Productions were calculated per manufacturer's instructions after subtracting the value of the “blank” wells to remove background absorbance values. The levels of PGI2 or TXA2 in NP-UAECs and P-UAECs are expressed as the fold or ratio increases over untreated control corrected against a standard curve, non-specific binding, maximum binding and normalized to the amount of protein per well.

Statistical Analysis

Data are presented as a fold change of untreated control and expressed as Mean ± SEM. For the PGI2/TXA2 ratio, data are presented as the ratio of PGI2/TXA2 calculated from the absolute pg/ml concentrations from the same treatment wells in duplicates from all cell lines studied. Data were analyzed using a two-way ANOVA (SigmaPlot 11 Statistical Software). When appropriate, an analysis of the simple effect was performed using one-way ANOVA followed by post hoc Student-Newman-Keuls test. Pairwise comparisons were performed using Student-Newman-Keuls test. Level of significance was established a priori at P<0.05.

Results

Expression of cPLA2, COX-1, PGIS and TXAS in NP-UAECs versus P-UAECs

Western immunoblotting revealed the protein expression of cPLA2, COX-1, PGIS and TXAS in both NP-UAECs and P-UAECs (Figure 1A). Densitometric analyses showed that the protein expressions of cPLA2, COX-1 and PGIS were significantly higher in P-UAECs compared to NP-UAECs (Figure 1B). In contrast, the expression of TXAS was significantly reduced in P-UAECs compared to NP-UAECs (Figure 1B).

Figure 1
Expression of the Prostanoid System Enzymes in NP-UAECs and P-UAECs

Basal and Calcium Ionophore Stimulated PGI2 and TXA2 Production in NP-UAECs and P-UAECs

Basal production of PGI2 was higher by P-UAECs compared to NP-UAECs (Figure 2A). Unstimulated basal production of PGI2 by P-UAECs at 8, 12 and 24 hours were1.31 ± 0.23, 1.43 ± 0.23 and 1.53 ±0.23 folds respectively corresponding to 47±0.75, 58±0.70 and 68±0.16 pg/ml respectively at the same time points. A23187 stimulated significant production of PGI2 in P-UAECs compared to NP-UAECs (Figure 2B). Maximum production by P-UAECs at 12 hours was 10.8 ± 0.39 fold of control compared to maximum production by NP-UAECs of 6.0 ± 0.37 fold of control.

Figure 2
Basal Unstimulated and Calcium Ionophore-Stimulated PGI2 and TXA2 Production in NP-UAECs and P-UAECs

Basal production of TXA2 by P-UAECs was not different compared to production by NP-UAECs (Figure 2C). Unstimulated basal production of TXA2 by P-UAECs at 8, 12 and 24 hours were 1.18 ± 0.19, 1.20 ± 0.19, 1.33 ± 0.36 folds respectively corresponding to 13±1.0, 20±1.1 and 23±0.22 pg/ml respectively at the same time points. However, A23187 stimulated significantly lower concentration-dependent production of TXA2 in P-UAECs compared to NP-UAECs (Figure 2D). The maximum production by P-UAECs at 12 hours was 1.49 ± 0.09 fold of control compared to maximum production by NP-UAECs at 1.93 ± 0.64 fold of control.

E2β, 2-OHE2, 4-OHE2, 2-ME2 and 4-ME2 Stimulated a Time and Concentration-Dependent PGI2 Increase in P-UAECs

Time- and concentration-dependent PGI2 increase was observed in P-UAECs after E2β treatment with highest responses observed at a concentration of 100 nmol/L at 12 hours treatment time (Figure 3A). Increase of PGI2 was noted after 4 hours in response to almost all concentration of E2β studied with the exception of 0.1 nmol/L. However, at 4, 8, 12, 24 hours, there were significant differences in PGI2 levels in response to 10 and 100 nmol/L E2β which were higher compared to 0.1 or 1 nmol/L concentrations.

Figure 3
Estradiol-17β and its Metabolites Induced Time and Concentration-Dependent Increases in Prostacyclin Production in P-UAECs

Similarly, exposure of P-UAECs to 2-OHE2 and 4-OHE2 treatments also stimulated a time and concentration-dependent PGI2 increase by P-UAECs with maximum responses observed at a concentration of 100 nmol/L at 12 hours treatment time (Figure 3B and 3C). Nevertheless, PGI2 increase was noted after 4 hours in response to all concentration of 2-OHE2 and 4-OHE2 studied and this increased in a time- and concentration-dependent manner compared to untreated control. No further increases in 2-OHE2 and 4-OHE2 -induced PGI2 increase by P-UAECs was seen after 12 hours at all concentrations studied. There were significant differences in PGI2 increase in response to 10 and 100 nmol/L of 2-OHE2 and 4-OHE2 which were higher compared to 0.1 or 1 nmol/L concentrations at 2, 4, 8, 12, 24 hours. However, 0.1 or 1 nmol/L concentrations stimulated significant increase of PGI2 compared to untreated control at 2, 4, 8, 12, 24 hours.

The time and concentration course of PGI2 levels in P-UAECs induced by 2-ME2 and 4-ME2 is shown in Figure 3D and 3E. Both methoxyestradiols increased PGI2 in a time- and concentration-dependent manner with maximal effects observed at a concentration of 100 nmol/L at 12 hours treatment time. However, increase of PGI2 by P-UAECs was seen at all concentrations studied at 2 hours of treatment time or greater. There were significant differences in PGI2 increase in response to 1, 10 and 100 nmol/L of 2-ME2 and 4-ME2 which were higher compared to 0.1nmol/L concentration at 4 hours of treatment time or greater.

E2β, 2-OHE2, 4-OHE2, 2-ME2 and 4-ME2 Stimulated a Greater Concentration-Dependent PGI2 Increase by P-UAECs more than NP-UAECs

A concentration-dependent increase in production of PGI2 was observed by P-UAECs after E2β treatment with highest responses observed at a concentration of 100 nmol/L (Figure 4A). On the other hand, E2β also induced PGI2 increases by NP-UAECs, however, the increase by NP-UAECs was significantly less compared to P-UAECs and no further concentration response was observed after the 10 nmol/L concentration (Figure 4A). Differences in PGI2 increases between NP-UAECs and P-UAECs were observed at 1 nmol/L, 10 nmol/L, 100 nmol/L and 1 μmol/L concentrations.

Figure 4
Estradiol-17β and its Metabolites Induced Concentration-Dependent Increases in Prostacyclin Production in P-UAECs compared to NP-UAECs

Similar to E2β, 2-OHE2 and 4-OHE2 treatments also stimulated greater concentration-dependent PGI2 production increases by P-UAECs compared to NP-UAECs (Figure 4B and 4C). Maximum responses were observed at a concentration of 100 nmol/L with no further increases seen with higher concentrations. 2-OHE2 and 4-OHE2 treatments did stimulate elevations of the production of PGI2 in NP-UAECs; however, these responses were significantly lower compared to the P-UAEC responses with no further increases seen after the 10 nmol/L concentration.

2-ME2 and 4-ME2 treatments also stimulated greater concentration-dependent PGI2 production increases by P-UAECs compared to NP-UAECs (Figure 4D and 4E. Maximum responses of P-UAECs to 2-ME2 was seen at 100 nmol/L with no further increases at greater doses. On the other hand, 4-ME2 induced maximum PGI2 production increases by P-UAECs at a concentration of 10 nmol/L with no significant further increases with higher concentrations. Interestingly, 4-ME2 was the only estrogen metabolite that stimulated a difference in PGI2 production responses between NP-UAECs and P-UAECs at a concentration as low as 0.1 nmol/L. PGI2 production was also noted by NP-UAECs in response to 2-ME2 and 4-ME2; however, these responses were of lower magnitude compared to P-UAEC responses with no further increases seen after the 10 nmol/L concentration.

The levels of TXA2 production stimulated by E2β, 2-OHE2, 4-OHE2, 2-ME2 and 4-ME2 increased linearly but did not reach statistical significance compared to untreated control in either NP-UAECs or P-UAECs. Therefore, these TXA2 productions are discussed within the context of the PGI2/TXA2 ratio.

The PGI2/TXA2 ratio: E2β, 2-OHE2, 4-OHE2, 2-ME2 and 4-ME2 Stimulated a Greater Concentration-Dependent PGI2/TXA2 ratio in P-UAECs more than NP-UAECs

As illustrated in the table 1, the basal PGI2/TXA2 ratio was higher in P-UAECs compared to NP-UAECs at 12 hrs treatment time. There was a concentration-dependent increase in the PGI2/TXA2 ratio when P-UAECs were exposed to E2β and its metabolites..

Table 1
The Effects of E2β, 2-OHE2, 4-OHE2, 2-ME2 and 4-ME2 on the PGI2/TXA2 Ratio in NP-UAECs versus P-UAECs.

E2β, 2-OHE2, 4-OHE2, 2-ME2 and 4-ME2 Stimulated de novo PGI2 Increase in P-UAECs via COX-1 and PGIS

Antagonism with the COX-1 inhibitor SC-560 and the PGIS inhibitor U-51605 were tested at all concentrations (0.1, 1, 10 and 100 nmol/L) of E2β and its metabolites studied, however since all concentration yielded similar results, only the responses from the optimal concentration of 100 nmol/L at 12 hrs treatment time are shown here. Neither SC-560 nor U-51605 (1μmol/L, 1hr) pretreatments significantly altered basal control PGI2 increase in P-UAECs (Figure 5A and 5B). However, both antagonists completely abrogated the PGI2 production in P-UAECs stimulated by 100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, 4-ME2 and the Ca2+ Ionophore (A23187)); (data not shown) indicating that the PGI2 production seen is de novo via the activities of COX-1 and PGIS (Figure 5A and 5B).

Figure 5
Estradiol-17β and its Metabolites Stimulated de novo PGI2 Production in P-UAECs via COX-1 and PGIS

E2β but Not its Metabolites Stimulated PGI2 Production in P-UAECs via the classic ERs

Antagonism with ICI 182,780 was tested at 0.1, 1, 10 and 100 nmol/L of E2β and its metabolites studied, however since all concentration yielded similar results, only the responses from the optimal concentration of 100 nmol/L are shown. Furthermore, since the NP-UAECs exhibited lower non-significant responses, antagonism studies were only carried out in P-UAECs. ICI 182,780 alone did not affect basal PGI2 production in P-UAECs; however, it totally abrogated PGI2 production in response to E2β, indicating possibility of involvement of either ER-α and/or ER-β (Figure 6). In contrast, ICI 182,780 did not have an effect on P-UAEC PGI2 production in response to2-OHE2, 4-OHE2, 2-ME2 or 4-ME2 indicating ER-independent mechanisms. ICI 182,780 did not have an effect on the PGI2 production of P-UAECs in response to nonreceptor stimulation with A23187 (data not shown) validating specific ER-mediated E2β responses (Figure 6).

Figure 6
Estradiol-17β but not its Metabolites Stimulated PGI2 Production in P-UAECs via the classic ERs

E2β but Not its Metabolites Stimulated PGI2 Production in P-UAECs Selectively via ER-α and Independent of ER-β

Similar to ICI 182,780 studies, antagonism with MPP and PHTPP were tested at all concentrations (0.1, 1, 10 and 100 nmol/L) of E2β and its metabolites studied; all concentrations examined yielded similar results, therefore only the data from the optimal concentration of 100 nmol/L are shown. In P-UAECs, ER-α blockade with 1 μmol/L of MPP completely abolished the PGI2 increase stimulated by 100 nmol/L of E2β indicating a role for ER-α (Figure 7A). In contrast, MPP did not abrogate the PGI2 production stimulated by 100 nmol/L of 2-OHE2, 4-OHE2, 2-ME2 or 4-ME2 similar to the above ICI 182,780 (Figure 7A). In contrast, E2β-induced PGI2 production was not inhibited by 1 μmol/L of the ER-β selective antagonist PHTPP demonstrating a lack of requirement for ER-β in these responses (Figure 7B). Similar to MPP, PHTPP did not did not inhibit the PGI2 production stimulated by 100 nmol/L of 2-OHE2, 4-OHE2, 2-ME2 or 4-ME2 (Figure 7B). Both MPP and PHTPP did not inhibit PGI2 production of P-UAECs induced by the nonreceptor stimulation using Ca2+ Ionophore (A23187); (data not shown) further validating specific ER-α mediated E2β responses.

Figure 7
Estradiol-17β but not its Metabolites Stimulated PGI2 Production in P-UAECs Selectively via ER-α and Independent of ER-β

Discussion

The key findings observed from this study are: (1) Pregnancy induces higher protein expression of cPLA2, COX-1 and PGIS in P-UAECs compared to NP-UAECs as well as a lower protein expression of TXAS in P-UAECs compared NP-UAECs; (2) E2β and its metabolites, 2-OHE2, 4-OHE2, 2-ME2 and 4-ME2, stimulate time and concentration-dependent increase in PGI2 in P-UAECs; (3) E2β and its metabolites stimulate a concentration-dependent increase in the PGI2/TXA2 ratio more in P-UAECs compared to NP-UAECs (4) E2β and its metabolites stimulate de novo PGI2 production in P-UAECs via activities of COX-1 and PGIS; and (5) E2β-induced PGI2 production in P-UAECs is mediated primarily via ER-α and independent of ER-β, whereas 2-OHE2, 4-OHE2, 2-ME2 and 4-ME2 stimulate PGI2 production in P-UAECs independent of either ER-α or ER-β.

We demonstrate herein that P-UAECs highly express the prostanoid system enzymes including cPLA2, COX-1 and PGIS. These data are consistent with reports of increased expression of these enzymes in ovine uterine arteries in vivo and ex vivo6, 13, 20, 21 and shows that the elevated expression during gestation are maintained to a great extent even through passaging in culture. However, in the present study we demonstrate, for the first time, that TXAS is expressed in UAECs and that expression is lower in P-UAECs compared to NP-UAECs. Taken together, the data from our studies suggest that during pregnancy, the prostanoid enzyme system shifts its expression pattern in the favor of more PGI2 production and less TXA2 production in support of rises and/or maintenance of uterine blood flow. These results demonstrate that P-UAEC PGI2 production is induced by gestational programming at the level of increased endothelial cell signaling, supporting earlier reports that pregnancy-specific programming in P-UAECs leads to increased basal and agonist-mediated responsiveness.6 The retention of programming in P-UAECs, perhaps via epigenetic mechanims, may also be responsible for elevated responses to steroids in subsequent pregnancies in vivo.22 We also show herein that the calcium ionophore (A23187) induces significant increases of PGI2 in P-UAECs and TXA2 in NP-UAECs demonstrating that the prostanoid enzymes expressed in these endothelial cells are functional and capable of eliciting calcium-dependent and receptor-independent PGI2 and TXA2 production. In this study, we demonstrate that E2β induces concentration-dependent increase of PGI2 and PGI2/TXA2 ratio more in P-UAECs than in NP-UAECs. Although maximum responses are noted at a high concentration of 100 nmol/L, low physiological concentrations of 1 and 10 nmol/L stimulated significant concentration-dependent increase of PGI2 and PGI2/TXA2 ratio more in P-UAECs than in NP-UAECs. In support of these findings are reports that E2β induces production of PGI2 in other cultured endothelial cells such as HUVECs, bovine pulmonary artery and aortic endothelial cells.14, 23-25 We have previously reported that the infusion of E2β in sheep significantly increases the ex vivo uterine arterial production of the stable PGI2 metabolite 6-keto-PGF1α.21 However, to the best of our knowledge, this is the first study to evaluate the in vitro comparison of E2β-induced endothelial PGI2 production and elevated PGI2/TXA2 ratio in the pregnant versus the nonpregnant states. In this regard, these data are also consistent with our previous findings that P-UAECs exhibit pregnancy-specific PGI2 production in response to ATP, basic fibroblast growth factor and epidermal growth factor.6

Our findings that 2-OHE2 and 4-OHE2 stimulate the increase of PGI2 and PGI2/TXA2 ratio more in P-UAECs than in NP-UAECs supports our hypothesis that CYP450- derived metabolites of E2β may play a role in the regulation of vascular responsiveness during pregnancy. It has been previously demonstrated that the uterine arterial infusion of 2-OHE2 and 4-OHE2 in sheep and gilts causes vasodilation and increase in uterine blood flow.26, 27 2-OHE2 and 4-OHE2 also significantly augment endothelial-dependent vasodilation of preconstricted vascular beds in ZSF1 rats, an animal model for hypertension, Type 2 diabetes, hyperlipidemia, nephropathy, metabolic syndrome.16, 28 Since 2-OHE2 and 4-OHE2 are rapidly converted to their methoxy derivatives in the presence of COMT, it is likely that the actions of the catecholestradiols on endothelial PGI2/TXA2 ratio maybe partly modulated by COMT expression and activity in these endothelial cells. Nevertheless, these observations suggest that the metabolism of E2β to the catecholestradiols, 2-OHE2 and 4-OHE2, mayalsoplay an essential role in the regulation of physiologic vascular responsiveness via production of endothelial-derived vasodilatory factors.

The observation that 2-ME2 and 4-ME2 enhance PGI2 levels and PGI2/TXA2 ratio in P-UAECs more than in NP-UAECs supports evidence that methoxyestradiols may positively influence vascular responsiveness during pregnancy. Consistent with these findings is the report that 2-ME2 induces PGI2 production in HUVECs.19 Low 2-ME2 level has been implicated in preeclampsia, a disease characterized by low plasma and urinary PGI2 and impaired uterine blood flow.29, 30 These observations suggest that 2-ME2 may be a promising physiological as well as pharmacological agent capable of clinically improving vascular responsiveness. We demonstrate for the first time that, 4-ME2 also stimulates PGI2 increases in vitro greater in the pregnant compared to the nonpregnant state and indeed may play role in positive pregnancy-induced uterine vascular responsiveness. Because 2-ME2 and 4-ME2 induced PGI2 increases at very low physiologic concentrations compared to E2β and the catecholestradiols suggests that the methoxyestradiols maybe more potent under these conditions and points to the notion that the vascular effects of locally produced and/or circulating estrogen metabolites may be more critical than previously thought compared to the vascular effects of the parent substrate.

Demonstrating a role for ER-α and/or ER-β, ICI which nonspecifically blocks ER-α and ER-β completely abrogated E2β-induced PGI2 production in P-UAECs. Previous studies have demonstrated that ICI also inhibits E2β-induced production of PGI2 in other cultured endothelial cells including HUVECs and ovine fetal pulmonary artery endothelial cells.14 However, because of the potential relevance of ER subtype selectivity in vascular function, there is considerable interest in investigating whether the classical ERs exhibit functional heterogeneity in the regulation of E2β-induced endothelial functions. Our data shows that E2β-induced PGI2 increases in P-UAECs is completely inhibited by the ER-α-specific MPP and unaffected by ER-β-specific PHTPP demonstrating that E2β-induced PGI2 production in P-UAECs is primarily mediated by ER-α. These data are in agreement with previous observations that PGI2 production by HUVECs was seen when these cells were treated with an ER-α selective agonist 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT).14 These data support the notions that whilst E2β binds and activates both ER-α and ER-β, it is the molecular and structural based differences in these receptors allow for a wide range of functional heterogeneity which may partly explain the selective actions of E2β.

In the present study, unlike E2β, the effects of its metabolites on PGI2 production in P-UAECs are not inhibited by the ER antagonists used in this study and thus may not be mediated via ER-α and/or ER-β. This is in agreement with several reports that the vascular physiology and pharmacology of E2β metabolites on many cell types including endothelial cells occur via ER-independent mechanisms.16-18 Thus other receptor-mediated mechanisms not involving the classical ERs may mediate E2β metabolites-induced PGI2 production in P-UAECs. Indeed we and others have reported that the genomic effects of E2β metabolites including proliferation of P-UAECs and suppression of pancreatic islet insulin release are mediated via the adrenergic receptors.18, 31 Nevertheless, the exact mechanism of action of E2β metabolites on non-genomic PGI2 increases in P-UAECs remains to be determined and may likely involve adrenergic receptors and/or other estrogen-associated receptors like G-protein-coupled receptor -30.

In conclusion, the findings from this current study demonstrates that the uterine endothelium exhibits pregnancy-specific increases in cPLA2, COX-1 and PGIS and decreases in TXAS shifting in the endothelial PGI2/TXA2 ratio toward more PGI2 production in association with rises and/or maintenance of uterine blood flow during pregnancy. Furthermore, we provide evidence that E2β primarily via ER-α and its metabolites via ER-independent mechanisms stimulate a higher de novo increase of the endothelial-derived vasodilator PGI2 and PGI2/TXA2 ratio in the pregnant compared to the nonpregnant state. Although maximum production of PGI2 and PGI2/TXA2 ratio was noted with the high concentration of 100 nmol/L, low physiologically relevant concentrations of 1 and 10 nmol/L of E2β and its metabolites also stimulated significant synthesis of these prostanoids more in P-UAECs compared to NP-UAECs. Collectively, the selective responses of P-UAECs further illustrate pregnancy specific programming at the level of the uterine artery endothelium signaling resulting in enhanced E2β and its metabolites-mediated induction of PGI2 synthesis in P-UAECs without significantly affecting TXA2 production.

Perspectives

The mechanisms by which estrogens regulate vascular tone and vasuclar responsiveness during pregnancy are not well understood. However, studies have shown that it likely involves ER-mediated stimulation of endothelial-derived vasodilatory factors including nitric oxide and/or PGI2.1,11 Herein, we demonstrate novel and compelling evidence that the vasoactive/vasoprotective effects of E2β during pregnancy may also involve its sequential conversion to catecholestradiols and methoxyestradiols which are capable of stimulating ER-independent endothelial PGI2 synthesis. Additional studies are required to understand whether the ER-independent induction of endothelial PGI2 synthesis by estrogen metabolites within uterine vasculature represents unappreciated signaling complexity of estrogens or just simply an evolutionary functional redundancy. Nevertheless, our findings necessitate the evaluation of catecholestradiols and methoxyestradiols in the regulation of vascular tone in physiology via endothelial-derived relaxing factors as well as dysregulation in the pathophysiology of vascular diseases such as hypertension, atherosclerosis and gestational vascular diseases such as preeclampsia.

Novelty and Significance

What Is New?

  • The uterine endothelium exhibits pregnancy-specific increases in phospholipase A2, cyclooxygenase-1 and prostacyclin synthase and decreases in endothelial thromboxane synthase expression.
  • Estradiol-17β primarily via estrogen receptor-α and its metabolites 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol and 4-methoxyestradiol via estrogen receptor-independent mechanisms stimulate higher de novo increase of the endothelial-derived prostacyclin/thromboxane ratio in the pregnant compared to the nonpregnant state.

What Is Relevant?

  • The selective pregnancy-specific endothelial prostanoid enzyme expressions and increase in prostacyclin/thromboxane ratio demonstrates roles for catecholestradiols and methoxyestradiols in the regulation of vascular responsiveness in physiology via endothelial-derived relaxing factors as well as in the pathophysiology of vascular diseases characterized by blunted endothelium-dependent vasodilator responses such as hypertension, atherosclerosis and preeclampsia.

Summary

  • These findings demonstrate novel and compelling evidence that the vasoactive/vasoprotective effects of estradiol-17β may involve its sequential conversion to catecholestradiols and methoxyestradiols which are capable of stimulating estrogen receptor-independent endothelial prostacyclin synthesis.

Acknowledgments

We thank members of the Perinatal Research Labs, University of Wisconsin-Madison and the Platelet and Neutrophil Immunology Lab, Blood Center of Wisconsin.

Sources of Funding: This work was supported by National Institutes of Health grants HL49210, HD38843, HL87144 (to RR Magness), AA19446 (to J Ramadoss), R25-GM083252 (to ML Carnes) and T32-HD041921-07 (to IM Bird).

Footnotes

Disclosures: None

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Félétou M, Huang Y, Vanhoutte PM. Endothelium-mediated control of vascular tone: Cox-1 and cox-2 products. Br J Pharmacol. 2011;164:894–912. [PMC free article] [PubMed]
2. Kobayashi T, Tahara Y, Matsumoto M, Iguchi M, Sano H, Murayama T, Arai H, Oida H, Yurugi-Kobayashi T, Yamashita JK, Katagiri H, Majima M, Yokode M, Kita T, Narumiya S. Roles of thromboxane a(2) and prostacyclin in the development of atherosclerosis in apoe-deficient mice. J Clin Invest. 2004;114:784–794. [PMC free article] [PubMed]
3. Egan KM, Lawson JA, Fries S, Koller B, Rader DJ, Smyth EM, Fitzgerald GA. Cox-2-derived prostacyclin confers atheroprotection on female mice. Science. 2004;306:1954–1957. [PubMed]
4. Mills JL, DerSimonian R, Raymond E, Morrow JD, Roberts LJ, Clemens JD, Hauth JC, Catalano P, Sibai B, Curet LB, Levine RJ. Prostacyclin and thromboxane changes predating clinical onset of preeclampsia: A multicenter prospective study. JAMA. 1999;282:356–362. [PubMed]
5. Magness RR, Shideman CR, Habermehl DA, Sullivan JA, Bird IM. Endothelial vasodilator production by uterine and systemic arteries. V. Effects of ovariectomy, the ovarian cycle, and pregnancy on prostacyclin synthase expression. Prostaglandins Other Lipid Mediat. 2000;60:103–118. [PubMed]
6. Bird IM, Sullivan JA, Di T, Cale JM, Zhang L, Zheng J, Magness RR. Pregnancy-dependent changes in cell signaling underlie changes in differential control of vasodilator production in uterine artery endothelial cells. Endocrinology. 2000;141:1107–1117. [PubMed]
7. Rupnow HL, Phernetton TM, Modrick ML, Wiltbank MC, Bird IM, Magness RR. Endothelial vasodilator production by uterine and systemic arteries. VIII. Estrogen and progesterone effects on CPLA2, COX-1, and PGIS protein expression. Biol Reprod. 2002;66:468–474. [PubMed]
8. Magness RR, Rosenfeld CR, Faucher DJ, Mitchell MD. Uterine prostaglandin production in ovine pregnancy: Effects of angiotensin ii and indomethacin. Am J Physiol. 1992;263:H188–197. [PubMed]
9. Albrecht ED, Pepe GJ. Placental steroid hormone biosynthesis in primate pregnancy. Endocr Rev. 1990;11:124–150. [PubMed]
10. Magness R. Maternal cardiovascular and other physiologic responses to the endocrinology of pregnancy. In: Bazer F, editor. The endocrinology of pregnancy. Totowa, NJ: Humana press Inc; 1998. pp. 507–539.
11. Magness RR, Phernetton TM, Gibson TC, Chen DB. Uterine blood flow responses to ICI 182 780 in ovariectomized oestradiol-17beta-treated, intact follicular and pregnant sheep. J Physiol. 2005;565:71–83. [PubMed]
12. Rosenfeld CR, Morriss FH, Battaglia FC, Makowski EL, Meschia G. Effect of estradiol-17beta on blood flow to reproductive and nonreproductive tissues in pregnant ewes. Am J Obstet Gynecol. 1976;124:618–629. [PubMed]
13. Rupnow HL, Phernetton TM, Modrick ML, Wiltbank MC, Bird IM, Magness RR. Endothelial vasodilator production by uterine and systemic arteries. Viii. Estrogen and progesterone effects on cpla2, cox-1, and pgis protein expression. Biol Reprod. 2002;66:468–474. [PubMed]
14. Sobrino A, Oviedo PJ, Novella S, Laguna-Fernandez A, Bueno C, García-Pérez MA, Tarín JJ, Cano A, Hermenegildo C. Estradiol selectively stimulates endothelial prostacyclin production through estrogen receptor-{alpha} J Mol Endocrinol. 2010;44:237–246. [PubMed]
15. Zhu BT, Conney AH. Functional role of estrogen metabolism in target cells: Review and perspectives. Carcinogenesis. 1998;19:1–27. [PubMed]
16. Dubey RK, Tofovic SP, Jackson EK. Cardiovascular pharmacology of estradiol metabolites. J Pharmacol Exp Ther. 2004;308:403–409. [PubMed]
17. Jobe SO, Ramadoss J, Koch JM, Jiang Y, Zheng J, Magness RR. Estradiol-17beta and its cytochrome p450- and catechol-o-methyltransferase-derived metabolites stimulate proliferation in uterine artery endothelial cells: Role of estrogen receptor-alpha versus estrogen receptor-beta. Hypertension. 2010;55:1005–1011. [PMC free article] [PubMed]
18. Jobe SO, Fling SN, Ramadoss J, Magness RR. A novel role for an endothelial adrenergic receptor system in mediating catecholestradiol-induced proliferation of uterine artery endothelial cells. Hypertension. 2011;58:874–881. [PMC free article] [PubMed]
19. Seeger H, Mueck AO, Lippert TH. Effect of estradiol metabolites on prostacyclin synthesis in human endothelial cell cultures. Life Sci. 1999;65:PL167–170. [PubMed]
20. Janowiak MA, Magness RR, Habermehl DA, Bird IM. Pregnancy increases ovine uterine artery endothelial cyclooxygenase-1 expression. Endocrinology. 1998;139:765–771. [PubMed]
21. Vagnoni KE, Magness RR. Estrogen and lipopolysaccharide stimulation of prostacyclin production and the levels of cyclooxygenase and nitric oxide synthase in ovine uterine arteries. Biol Reprod. 1998;59:1008–1015. [PubMed]
22. Dasgupta C, Chen M, Zhang H, Yang S, Zhang L. Chronic hypoxia during gestation causes epigenetic repression of the estrogen receptor-α gene in ovine uterine arteries via heightened promoter methylation. Hypertension. 2012;60:697–704. [PMC free article] [PubMed]
23. Madden MC, Vender RL, Friedman M. Effect of hypoxia on prostacyclin production in cultured pulmonary artery endothelium. Prostaglandins. 1986;31:1049–1062. [PubMed]
24. Nawroth PP, Stern DM, Kaplan KL, Nossel HL. Prostacyclin production by perturbed bovine aortic endothelial cells in culture. Blood. 1984;64:801–806. [PubMed]
25. Oriji GK. Endothelin-induced prostacyclin production in rat aortic endothelial cells is mediated by protein kinase c. Prostaglandins Leukot Essent Fatty Acids. 1999;60:263–268. [PubMed]
26. Rosenfeld CR, Jackson GM. Induction and inhibition of uterine vasodilation by catechol estrogen in oophorectomized, nonpregnant ewes. Endocrinology. 1982;110:1333–1339. [PubMed]
27. Stice SL, Ford SP, Rosazza JP, Van Orden DE. Interaction of 4-hydroxylated estradiol and potential-sensitive ca2+ channels in altering uterine blood flow during the estrous cycle and early pregnancy in gilts. Biol Reprod. 1987;36:369–375. [PubMed]
28. Tofovic SP, Dubey RK, Jackson EK. 2-hydroxyestradiol attenuates the development of obesity, the metabolic syndrome, and vascular and renal dysfunction in obese zsf1 rats. J Pharmacol Exp Ther. 2001;299:973–977. [PubMed]
29. Kanasaki K, Palmsten K, Sugimoto H, Ahmad S, Hamano Y, Xie L, Parry S, Augustin HG, Gattone VH, Folkman J, Strauss JF, Kalluri R. Deficiency in catechol-o-methyltransferase and 2-methoxyoestradiol is associated with pre-eclampsia. Nature. 2008;453:1117–1121. [PubMed]
30. Svedas E, Nisell H, Vanwijk MJ, Nikas Y, Kublickiene KR. Endothelial dysfunction in uterine circulation in preeclampsia: Can estrogens improve it? Am J Obstet Gynecol. 2002;187:1608–1616. [PubMed]
31. Etchegoyen GS, Cardinali DP, Pérez AE, Tamayo J, Pérez-Palacios G. Binding and effects of catecholestrogens on adenylate cyclase activity, and adrenoceptors, benzodiazepine and gaba receptors in guinea-pig hypothalamic membranes. Eur J Pharmacol. 1986;129:1–10. [PubMed]