Abstract-Estradiol-17␤ (E 2 ␤) and its metabolites, which are sequentially synthesized by cytochrome P450s and catechol-O-methyltransferase to form 2 and 4-hydroxyestradiol (OHE 2 ) and 2-and 4-methoxestradiol (ME 2 ), are elevated during pregnancy. We investigated whether cytochrome P450s and catechol-O-methyltransferase are expressed in uterine artery endothelial cells (UAECs) and whether E 2 ␤ and its metabolites modulate cell proliferation via ER-␣ and/or ER-␤ and play roles in physiological uterine angiogenesis during pregnancy. Cultured ovine UAECs from pregnant and nonpregnant ewes were treated with 0.1 to 100.0 nmol/L of E 2 ␤, 2-OHE 2 , 4-OHE 2 , 2-ME 2 , and 4-ME 2 . ER-␣ or ER-␤ specificity was tested using ICI 182 780, ER-␣-specific 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinyleth oxy)phenol]-1H-pyrazole dihydrochloride, ER-␤-specific 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo [1,5-a]pyrim idin-3-yl]phenol antagonists and their respective agonists ER-␣-specific 4,4Ј,4Љ-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and ER-␤-specific 2,3-bis(4-Hydroxyphenyl)-propionitrile. Angiogenesis was evaluated using 5-bromodeoxyuridine proliferation assay. Using confocal microscopy and Western analyses to determine enzyme location and levels, we observed CYP1A1, CYP1A2, CYP1B1, CYP3A4, and catechol-O-methyltransferase expression in UAECs; however, expressions were similar between nonpregnant UAECs and pregnant UAECs. E 2 ␤, 2-OHE 2 , 4-OHE 2 , and 4-ME 2 treatments concentration-dependently stimulated proliferation in pregnant UAECs but not in nonpregnant UAECs; 2-ME 2 did not stimulate proliferation in either cell type. Proliferative responses of pregnant UAECs to E 2 ␤ were solely mediated by ER-␤, whereas responses to E 2 ␤ metabolites were neither ER-␣ nor ER-␤ mediated. We demonstrate an important vascular role for E 2 ␤, its cytochrome P450-and catechol-O-methyltransferasederived metabolites, and ER-␤ in uterine angiogenesis regulation during pregnancy that may be dysfunctional in preeclampsia and other cardiovascular disorders. (Hypertension. 2010;55:1005-1011.)Key Words: angiogenesis Ⅲ hypertension Ⅲ pregnancy Ⅲ endothelium Ⅲ estradiol metabolites Ⅲ CYP450s P regnancy is associated with dramatic uterine blood flow rises resulting from vascular adaptations, including vasodilatation and angiogenesis. 1 These adaptations are critical in pregnancy because their dysfunctions are implicated in pathological pregnancies, such as preeclampsia, which complicates 6% to 8% of all pregnancies in the United States and accounts for 50 000 maternal deaths per year worldwide. [2][3][4] Regulation of vascular adaptations during pregnancy is mediated partly by estrogens, which are elevated during gestation. 5 Estradiol-17␤ (E 2 ␤) infusion in sheep markedly reduces uterine and systemic vascular resistance causing rises in uterine and systemic blood flows. 6 Uterine arterial administration of the nonselective estrogen receptor (ER) antagonist ICI 182 780 (ICI) in pregnant sheep lowers uterine blood flow, demonstra...

Estrogens and estrogen metabolites have important functions in cardiovascular and other physiology, yet the patterns of estrogen synthesis, metabolism and the individual plasma profile of estrogens and estrogen metabolites during human pregnancy as well as in preeclampsia remain undetermined. We performed liquid chromatography mass spectrometry on plasma samples from normotensive pregnant women (normP; n = 8), women with mild (mPE; n = 8) and severe (sPE; n = 8) preeclampsia at labor. Compared to normP, estrone was lower in sPE, whereas plasma level of estradiol-17β was significantly lower in women with mPE and sPE. Estriol was lower in sPE but not in mPE. Although, 2-hydroxyestrone was lower in mPE and sPE, 4-hydroxyestrone was high in sPE. 16-α-hydroxyestrone was higher in mPE but not in sPE. 2-hydroxyestradiol in women with mPE and sPE were lower compared to normP. Compared to 2-methoxyestrone in normP, levels were lower in sPE. 3-methoxyestrone and 4-methoxyestrone were unchanged. 2-methoxyestradiol was lower in mPE and sPE; however, 4-methoxyestradiol was low only in sPE. Compared to normP, 16-keto-estradiol levels were significantly higher in sPE whereas 16-epi-estriol and 17-epi-estriol were lower in women with sPE. Our findings show that preeclampsia is characterized by aberrant synthesis, metabolism and accumulation of estrogens and estrogen metabolites that are likely to be associated with alterations in vascular function. These results underscore the need to investigate the functional vascular and other physiology of estrogens and estrogen metabolites in the pathophysiology of preeclampsia.

The steroid hormone estrogen and its classical estrogen receptors (ERs), ER-α and ER-β, have been shown to be partly responsible for the short- and long-term uterine endothelial adaptations during pregnancy. The ER-subtype molecular and structural differences coupled with the differential effects of estrogen in target cells and tissues suggest a substantial functional heterogeneity of the ERs in estrogen signaling. In this review we discuss (1) the role of estrogen and ERs in cardiovascular adaptations during pregnancy, (2) in vivo and in vitro expression of ERs in uterine artery endothelium during the ovarian cycle and pregnancy, contrasting reproductive and nonreproductive arterial endothelia, (3) the structural basis for functional diversity of the ERs and estrogen subtype selectivity, (4) the role of estrogen and ERs on genomic responses of uterine artery endothelial cells, and (5) the role of estrogen and ERs on nongenomic responses in uterine artery endothelia. These topics integrate current knowledge of this very rapidly expanding scientific field with diverse interpretations and hypotheses regarding the estrogenic effects that are mediated by either or both ERs and their relationship with vasodilatory and angiogenic vascular adaptations required for modulating the dramatic physiological rises in uteroplacental perfusion observed during normal pregnancy.

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.

Sequential conversion of estradiol-17β to its biologically active catecholestradiols 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) contributes importantly to its angiogenic effects on uterine artery endothelial cells derived from pregnant (P-UAECs), but not nonpregnant (NP-UAECs) ewes via estrogen receptor-independent mechanism. Because catecholestradiols and catecholamines exhibit structural similarities and have high affinity for α- and β-adrenergic receptors (ARs), we investigated if the endothelial α- or β-ARs mediate catecholestradiols-induced proliferation of P-UAECs and whether catecholamines alter these responses. Western analyses revealed expression of specific AR subtypes in NP-UAECs and P-UAECs including α2-, β2- and β3-ARs; not α1- and β1-ARs. Levels of β2-ARs and β3-ARs were unaltered by pregnancy; whereas α2-ARs were decreased. Norepinephrine and epinephrine increased P-UAEC, but not NP-UAEC proliferation and these effects were suppressed by propranolol (β-AR blocker) but not phentolamine (α-AR blocker). Catecholamines combinations with 2-OHE2 or 4-OHE2 enhanced P-UAEC mitogenesis. Catecholestradiol-induced P-UAECs proliferation was also inhibited by propranolol but not phentolamine. β2-AR and β3-AR antagonists (ICI 118,551and SR 59230A respectively) abrogated the mitogenic effects of both 2-OHE2 and 4-OHE2. Stimulation of β2-ARs and β3-ARs using Formoterol and BRL37344 dose-dependently stimulated P-UAEC proliferation which was abrogated by ICI 118,551and SR 59230A, respectively. Proliferation effects of both catecholamines and catecholestradiols were only observed in P-UAEC (not NP-UAEC) and were mediated via β2-ARs and β3-ARs. We demonstrate for the first time convergence of the endothelial AR and estrogenic systems in the regulating endothelial proliferation, thus providing a distinct evolutionary advantage for modulating uterine perfusion during stressful pregnancies.

Background Pregnancy-induced utero-placental growth, angiogenic remodeling, and enhanced vasodilation are all partly regulated by estradiol-17β-mediated activation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. However, very little is known about the effects of alcohol on these maternal utero-placental vascular adaptations during pregnancy and its potential role in the pathogenesis of Fetal Alcohol Spectrum Disorders (FASD). In this study, we hypothesized that in vitro chronic binge-like alcohol will decrease uterine arterial endothelial eNOS expression and alter its multi-site phosphorylation activity state via disruption of AKT signaling. To study the direct effects of alcohol on uterine vascular adaptations, we further investigated the effects of alcohol on estradiol-17β-induced uterine angiogenesis in vitro. Methods Uterine artery endothelial cells were isolated from pregnant ewes (gestational day 120-130; term = 147), Fluorescence Activated Cell sorted, validated, and maintained in culture to passage 4. To mimic maternal binge drinking patterns, cells were cultured in the absence or presence of a lower (LD) or higher dose (HD) of alcohol in a compensating sealed humidified chamber system equilibrated with aqueous alcohol for 3 h on 3 consecutive days. Immunoblotting was performed to assess expression of NO system-associated proteins and eNOS multi-site phosphorylation. Following this treatment paradigm, control and binge alcohol treated cells were passaged, grown for two days, and then treated with increasing concentrations of estradiol-17β (0.1, 1, 10, 100 nM) in the absence or presence of LD or HD alcohol to evaluate estradiol-17β-induced angiogenesis index using BrdU Proliferation Assay. Results LD and HD binge-like alcohol decreased uterine arterial eNOS expression (P=0.009). eNOS multi-site phosphorylation activation state was altered: P635eNOS was decreased (P=0.017), P1177 eNOS was not altered, and P495 eNOS exhibited an inverse U shaped dose-dependent relationship with alcohol. LD and HD alcohol decreased the major eNOS-associated protein cav-1 (P<0.001). However, the commonly implicated AKT pathway did not correlate with eNOS post-translational modifications. Assessment of uterine vascular adaptation via angiogenesis demonstrated that alcohol abrogated the dose-dependent proliferative effects of estradiol-17β and thus blunted angiogenesis. Conclusions Thus, the maternal uterine vasculature during pregnancy may be vulnerable to chronic binge-like alcohol. Altered eNOS multi-site phosphorylation also suggests that alcohol produces specific effects at the level of post-translational modifications critical for pregnancy-induced uterine vascular adaptations. Finally, the alcohol and estradiol-17β data suggest a negative impact of alcohol on estrogen actions on the uterine vasculature.

Estradiol 17β (E2β) and ascorbic acid (AA) have been implicated in cancer progression. However, little is known about the actions of biologically active metabolites of E2β, 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME2), and 4-methoxyestradiol (4ME2) synthesized sequentially by cytochrome P450, family 1, subfamily A (CYP1A1) and B (CYP1B1), polypeptide 1, and catechol-O-methyltransferase (COMT) on ovarian cancer. Herein, we examined the expression of CYP1A1, CYP1B1, COMT, and estrogen receptor α (ERα) and β (ERβ) in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, and OVCA-432). We also investigated the roles of E2β, 2OHE2, 4OHE2, 2ME2, and 4ME2 in cell proliferation, and their interactive effects with AA on ovarian cells. We found the expression of CYP1A1, CYP1B1, COMT, ERα, and ERβ in most cell lines tested. Treating cells with physiological concentrations of E2β and its metabolites promoted (13%-42% of the control) IOSE-385 and OVCAR-3 proliferation. The ER blockade inhibited IOSE-385 (∼76%) and OVCAR-3 (∼87%) proliferative response to E2β but not to its metabolites. The ERα blockade inhibited (∼85%) E2β-stimulated OVCAR-3 proliferation, whereas ERβ blockade attenuated (∼83%) E2β-stimulated IOSE-385 proliferation. The AA at ≥250 μmol/L completely inhibited serum-stimulated cell proliferation in all cell lines tested; however, such inhibition in IOSE-385, OVCAR-3, and OVCA-432 was partially (∼10%-20%) countered by E2β and its metabolites. Thus, our findings indicate that E2β and its metabolites promote cell proliferation and antagonize the AA-suppressed cell proliferation in a subset of ovarian cancer cells, suggesting that blocking the actions of E2β and its metabolites may enhance AA's antiovarian cancer activity.

Previously we demonstrated that the biologically active metabolites of 17β-oestradiol, 2-hydroxyoestradiol (2-OHE ) and 4-hydroxyoestradiol (4-OHE ), stimulate pregnancy-specific proliferation of uterine artery endothelial cells derived from pregnant (P-UAECs), but not non-pregnant ewes. However, unlike 17β-oestradiol, which induces proliferation via oestrogen receptor-β (ER-β), the catecholoestradiols mediate P-UAEC proliferation via β-adrenoceptors (β-AR) and independently of classic oestrogen receptors. Herein, we aim to further elucidate the signalling mechanisms involved in proliferation induced by catecholoestradiols in P-UAECs. P-UAECs were treated with 2-OHE and 4-OHE for 0, 0.25, 0.5, 1, 2, 4, 12 and 24 h, to analyse activation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)-AKT. Specific inhibitors for ERK1/2 MAPK (PD98059), p38 MAPK (SB203580), JNK MAPK (SP600125), or PI3K (LY294002) were used to determine the involvement of individual kinases in agonist-induced P-UAEC proliferation. 2-OHE and 4-OHE stimulated biphasic phosphorylation of ERK1/2, slow p38 and JNK phosphorylation over time, and rapid monophasic AKT phosphorylation. Furthermore, ERK1/2, p38 and JNK MAPKs, but not PI3K, were individually necessary for catecholoestradiol-induced proliferation. In addition, when comparing the signalling mechanisms of the catecholoestradiols, to 17β-oestradiol and catecholamines, we observed that convergent MAPKs signalling pathways facilitate P-UAEC proliferation induced by all of these hormones. Thus, all three members of the MAPK family mediate the mitogenic effects of catecholoestradiols in the endothelium during pregnancy. Furthermore, the convergent signalling of MAPKs involved in catecholoestradiol-, 17β-oestradiol- and catecholamine-induced endothelial cell proliferation may be indicative of unappreciated evolutionary functional redundancy to facilitate angiogenesis and ensure maintenance of uterine blood flow during pregnancy.