Sheikh O. Jobe

Aberrant Synthesis, Metabolism, and Plasma Accumulation of Circulating Estrogens and Estrogen Metabolites in Preeclampsia: Implications for Vascular Dysfunction

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 with normP, estrone was lower in sPE, whereas plasma level of estradiol-17&bgr; 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-&agr;-hydroxyestrone was higher in mPE, but not in sPE. 2-hydroxyestradiol in women with mPE and sPE were lower compared with normP. Compared with 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 with normP, 16-keto-estradiol-17&bgr; 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.

Estradiol-17β and Its Cytochrome P450- and Catechol-O-Methyltransferase–Derived Metabolites Stimulate Proliferation in Uterine Artery Endothelial Cells. Role of Estrogen Receptor-α Versus Estrogen Receptor-β

Estradiol-17β and its metabolites which are sequentially synthesized by cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) to form 2 and 4-Hydroxyestradiol (2-OHE2 and 4-OHE2) and 2- and 4-Methoxestradiol (2-ME2, and 4-ME2) are elevated during pregnancy. We investigated whether CYP450s and COMT are expressed in uterine artery endothelial cells (UAECs) and if E2β and its metabolites modulate cell proliferation via ER-α and/or ER-β and play roles in physiologic uterine angiogenesis during pregnancy. Cultured ovine UAECs from pregnant (P-UAECs) and nonpregnant (NP-UAECs) ewes were treated with 0.1-100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, and 4-ME2. ER-α or ER-β specificity was tested using ICI 182,780, ER-α-specific MPP, ER-β –specific PHTPP antagonists and their respective agonists ER-α-specific PPT and ER-β –specific DPN. Angiogenesis was evaluated using BrdU Proliferation Assay. Utilizing confocal microscopy and Western analyses to determine enzyme location and levels, we observed CYP1A1, CYP1A2, CYP1B1, CYP3A4 and COMT expression in UAECs; however, expressions were similar between NP-UAECs and P-UAECs. E2β, 2-OHE2, 4-OHE2, and 4-ME2 treatments concentration-dependently stimulated proliferation in P-UAECs, but not NP-UAECs; 2-ME2 did not stimulate proliferation in either cell type. Proliferative responses of P-UAECs to E2β were solely mediated by ER-β, whereas responses to E2β metabolites were neither ER-α nor ER-β mediated. We demonstrate an important vascular role for E2β, its CYP450- and COMT-derived metabolites and ER-β in uterine angiogenesis regulation during pregnancy that may be dysfunctional in preeclampsia and other cardiovascular disorders.

Aberrant Synthesis, Metabolism, and Plasma Accumulation of Circulating Estrogens and Estrogen Metabolites in Preeclampsia

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 with normP, estrone was lower in sPE, whereas plasma level of estradiol-17&bgr; 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-&agr;-hydroxyestrone was higher in mPE, but not in sPE. 2-hydroxyestradiol in women with mPE and sPE were lower compared with normP. Compared with 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 with normP, 16-keto-estradiol-17&bgr; 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.

Estrogen Receptor-α and Estrogen Receptor-β in the Uterine Vascular Endothelium during Pregnancy: Functional Implications for Regulating Uterine Blood Flow

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.

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.

Estradiol-17β and its metabolites which are sequentially synthesized by cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) to form 2 and 4-Hydroxyestradiol (2-OHE2 and 4-OHE2) and 2- and 4-Methoxestradiol (2-ME2, and 4-ME2) are elevated during pregnancy. We investigated whether CYP450s and COMT are expressed in uterine artery endothelial cells (UAECs) and if E2β and its metabolites modulate cell proliferation via ER-α and/or ER-β and play roles in physiologic uterine angiogenesis during pregnancy. Cultured ovine UAECs from pregnant (P-UAECs) and nonpregnant (NP-UAECs) ewes were treated with 0.1-100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, and 4-ME2. ER-α or ER-β specificity was tested using ICI 182,780, ER-α-specific MPP, ER-β –specific PHTPP antagonists and their respective agonists ER-α-specific PPT and ER-β –specific DPN. Angiogenesis was evaluated using BrdU Proliferation Assay. Utilizing confocal microscopy and Western analyses to determine enzyme location and levels, we observed CYP1A1, CYP1A2, CYP1B1, CYP3A4 and COMT expression in UAECs; however, expressions were similar between NP-UAECs and P-UAECs. E2β, 2-OHE2, 4-OHE2, and 4-ME2 treatments concentration-dependently stimulated proliferation in P-UAECs, but not NP-UAECs; 2-ME2 did not stimulate proliferation in either cell type. Proliferative responses of P-UAECs to E2β were solely mediated by ER-β, whereas responses to E2β metabolites were neither ER-α nor ER-β mediated. We demonstrate an important vascular role for E2β, its CYP450- and COMT-derived metabolites and ER-β in uterine angiogenesis regulation during pregnancy that may be dysfunctional in preeclampsia and other cardiovascular disorders.

Estradiol-17β and its Cytochrome P450- and Catechol-O-Methyltransferase–Derived Metabolites Selectively Stimulate Production of Prostacyclin in Uterine Artery Endothelial Cells Role of Estrogen Receptor-α Versus Estrogen Receptor-β

Metabolism of estradiol-17&bgr; to 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 4-methoxyestradiol contributes importantly to the vascular effects of estradiol-17&bgr; in several vascular beds. However, little is known about the role of estradiol-17&bgr; metabolites via the different estrogen receptors (ER-&agr;/ER-&bgr;) on de novo endothelial prostacyclin and thromboxane production. We hypothesized that estradiol-17&bgr; and its metabolites, via ER-&agr; or ER-&bgr;, can enhance the prostacyclin/thromboxane ratio through the classic phospholipase A2, cyclooxygenase-1, and prostacyclin synthase pathway in ovine uterine artery endothelial cells (UAECs) derived from pregnant (P-UAECs) versus nonpregnant (NP-UAECs) ewes. Western analyses showed higher expression of phospholipase A2, cyclooxygenase-1, and prostacyclin synthase in UAECs from the pregnant state, whereas thromboxane synthase was lowered in UAECs from the pregnant state. In UAECs from the pregnant state, estradiol-17&bgr;, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol and 4-methoxyestradiol concentration and time-dependently increased prostacyclin compared with controls. Prostacyclin increases in UAECs from the nonpregnant state were of a lower magnitude. Estradiol-17&bgr; and its metabolites stimulated higher prostacyclin/thromboxane ratios in UAECs from the pregnant state compared with UAECs from the nonpregnant state. Estradiol-17&bgr;–induced prostacyclin increases were abrogated by the antagonists SC-560 (cyclooxygenase-1), U-51605 (Prostacyclin synthase), ICI 182 780 (ICI; both ER-&agr;/&bgr;), and 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinyleth oxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-&agr;), but not by 4-[2-phenyl-5,7-bis (trifluoromethyl) pyrazolo[1,5-a]pyrim idin-3-yl]phenol (PHTPP; ER-&bgr;). Prostacyclin increases induced by its metabolites were abolished by SC-560 and U-51605, but unaltered by ICI, MPP, or PHTPP. Our findings demonstrate that estrogen via primarily ER-&agr; 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 ERs.

A Novel Role for an Endothelial Adrenergic Receptor System in Mediating Catecholestradiol-Induced Proliferation of Uterine Artery Endothelial Cells

Sequential conversion of estradiol-17&bgr; to its biologically active catecholestradiols, 2-hydroxyestradiol (OHE2) and 4-OHE2, contributes importantly to its angiogenic effects on uterine artery endothelial cells (UAECs) derived from pregnant, but not nonpregnant ewes via an estrogen receptor-independent mechanism. Because catecholestradiols and catecholamines exhibit structural similarities and have high affinity for &agr;- and &bgr;-adrenergic receptors (ARs), we investigated whether the endothelial &agr;- or &bgr;-ARs mediate catecholestradiol-induced proliferation of P-UAECs and whether catecholamines alter these responses. Western analyses revealed expression of specific AR subtypes in nonpregnant UAECs and P-UAECs, including &agr;2-, &bgr;2-, and &bgr;3-ARs but not &agr;1- and &bgr;1-ARs. Levels of &bgr;2-ARs and &bgr;3-ARs were unaltered by pregnancy, whereas &agr;2-ARs were decreased. Norepinephrine and epinephrine increased P-UAEC, but not nonpregnant UAEC proliferation, and these effects were suppressed by propranolol (&bgr;-AR blocker) but not phentolamine (&agr;-AR blocker). Catecholamines combinations with 2-OHE2 or 4-OHE2 enhanced P-UAEC mitogenesis. Catecholestradiol-induced P-UAEC proliferation was also inhibited by propranolol but not phentolamine. &bgr;2-AR and &bgr;3-AR antagonists (ICI 118 551and SR 59230A, respectively) abrogated the mitogenic effects of both 2-OHE2 and 4-OHE2. Stimulation of &bgr;2-ARs and &bgr;3-ARs using formoterol and BRL 37344 dose-dependently stimulated P-UAEC proliferation, which was abrogated by ICI 118 551 and SR 59230A, respectively. Proliferation effects of both catecholamines and catecholestradiols were only observed in P-UAECs (not nonpregnant UAECs) and were mediated via &bgr;2-ARs and &bgr;3-ARs. We demonstrate for the first time convergence of the endothelial AR and estrogenic systems in regulating endothelial proliferation, thus providing a distinct evolutionary advantage for modulating uterine perfusion during stressful pregnancies.

Alcohol and maternal uterine vascular adaptations during pregnancy-part I: effects of chronic in vitro binge-like alcohol on uterine endothelial nitric oxide system and function.

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 (FASDs). In this study, we hypothesized that in vitro chronic binge-like alcohol will decrease uterine arterial endothelial eNOS expression and alter its multisite 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 to 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 hours 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 2 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 multisite phosphorylation activation state was altered: P(635) eNOS was decreased (p = 0.017), P(1177) eNOS was not altered, and P(495) 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 posttranslational 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 multisite phosphorylation also suggests that alcohol produces specific effects at the level of posttranslational 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 and Its Metabolites Stimulate Cell Proliferation and Antagonize Ascorbic Acid-Suppressed Cell Proliferation in Human Ovarian Cancer Cells

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.

Estradiol-17β and Its Cytochrome P450- and Catechol-O-Methyltransferase–Derived Metabolites Stimulate Proliferation in Uterine Artery Endothelial Cells

Estradiol-17β and its metabolites which are sequentially synthesized by cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) to form 2 and 4-Hydroxyestradiol (2-OHE2 and 4-OHE2) and 2- and 4-Methoxestradiol (2-ME2, and 4-ME2) are elevated during pregnancy. We investigated whether CYP450s and COMT are expressed in uterine artery endothelial cells (UAECs) and if E2β and its metabolites modulate cell proliferation via ER-α and/or ER-β and play roles in physiologic uterine angiogenesis during pregnancy. Cultured ovine UAECs from pregnant (P-UAECs) and nonpregnant (NP-UAECs) ewes were treated with 0.1-100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, and 4-ME2. ER-α or ER-β specificity was tested using ICI 182,780, ER-α-specific MPP, ER-β –specific PHTPP antagonists and their respective agonists ER-α-specific PPT and ER-β –specific DPN. Angiogenesis was evaluated using BrdU Proliferation Assay. Utilizing confocal microscopy and Western analyses to determine enzyme location and levels, we observed CYP1A1, CYP1A2, CYP1B1, CYP3A4 and COMT expression in UAECs; however, expressions were similar between NP-UAECs and P-UAECs. E2β, 2-OHE2, 4-OHE2, and 4-ME2 treatments concentration-dependently stimulated proliferation in P-UAECs, but not NP-UAECs; 2-ME2 did not stimulate proliferation in either cell type. Proliferative responses of P-UAECs to E2β were solely mediated by ER-β, whereas responses to E2β metabolites were neither ER-α nor ER-β mediated. We demonstrate an important vascular role for E2β, its CYP450- and COMT-derived metabolites and ER-β in uterine angiogenesis regulation during pregnancy that may be dysfunctional in preeclampsia and other cardiovascular disorders.