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Madison, WI, United States

Koch J.M.,Perinatal Research Laboratories | Ramadoss J.,University of Wisconsin - Madison | Magness R.R.,University of Wisconsin - Madison
Journal of Proteome Research | Year: 2010

Embryonic development is a time-sensitive period that requires a synchronized uterine environment, which is created by the secretion of proteins from both the embryo and uterus. Numerous studies have identified uterine luminal proteins and related these to specific adaptations during early pregnancy (EP). However, no study has yet utilized LC-MS/MS to identify the signature profile of proteins in the uterine lumen during EP. In this study, uterine luminal fluid from nonpregnant (NP; n = 3) and EP (n = 3; gestational day 16) ewes were analyzed by LC-MS/MS and validated by Western immunoblotting. We identified a unique signature profile for EP luminal fluid; 15 proteins related to specific aspects of embryonic development including growth and remodeling, immune system regulation, oxidative stress balance, and nutrition were significantly altered (up to 65-fold of NP) in EP profile. Specific uterine remodeling proteins such as transgelin (P = 0.008) and placental proteins like PP9 (P = 0.02) were present in EP luminal fluid but were barely detectable in the NP flushings. Direct correlations (R2 = 0.84, P = 0.01) were observed between proteomics and immunoblotting. These data provide information on dynamic physiological processes associated with EP at the level of the uterus and conceptus and may potentially demonstrate a signature profile associated with embryonic well-being. © 2010 American Chemical Society. Source

Jobe S.O.,University of Wisconsin - Madison | Tyler C.T.,University of Wisconsin - Madison | Magness R.R.,University of Wisconsin - Madison | Magness R.R.,Perinatal Research Laboratories
Hypertension | Year: 2013

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-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 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-17β 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. © 2012 American Heart Association, Inc. Source

Ramadoss J.,University of Texas Medical Branch | Magness R.R.,Perinatal Research Laboratories
Physiological Genomics | Year: 2012

Genomic studies on fetal alcohol spectrum disorders (FASD) have utilized either genome-wide microarrays/bioinformatics or targeted real-time PCR (RT-PCR). We utilized herein for the first time a novel digital approach with high throughput as well as the capability to focus on one physiological system. The aim of the present study was to investigate alcohol-induced alterations in uterine angiogenesisrelated mRNA abundance using digital mRNA technology. Four biological and three technical replicates of uterine arterial endothelial cells from third-trimester ewes were fluorescence-activated cell sorted, validated, and treated without or with binge-like alcohol. A capture probe covalently bound to an oligonucleotide containing biotin and a color-coded reporter probe were designed for 85 angiogenesis-related genes and analyzed with the Nanostring nCounter system. Twenty genes were downregulated (↓) and two upregulated (↑), including angiogenic growth factors/receptors (↓ placental growth factor), adhesion molecules (↓ angiopoietin-like-3; ↓ collagen-18A1; ↓ endoglin), proteases/matrix proteins/inhibitors (↓ alanyl aminopeptidase; ↓ collagen-4A3; ↓ heparanase; ↓ plasminogen, ↑ plasminogen activator urokinase; ↓ platelet factor-4; ↓ plexin domain containing-1; ↓ tissue inhibitor of metalloproteinases-3), transcription/signaling molecules (↓ heart and neural crest derivatives-2; ↓ DNA-binding protein inhibitor; ↓ NOTCH-4; ↓ ribosomal protein L13a1; ↓ ribosomal protein large-P1), cytokines/chemokines (↓ interleukin-1B), and miscellaneous growth factors (↓ leptin; ↓ plateletderived growth factor-α); ↓ transforming growth factor (TGF-α; ↑ TGF-β receptor-1). These novel data show significant detrimental alcohol effects on genes controlling angiogenesis supporting a mechanistic role for abnormal uteroplacental vascular development in FASD. The tripartite digital gene expression system is therefore a valuable tool to answer many additional questions about FASD from both mechanistic as well as ameliorative perspectives. © 2012 the American Physiological Society. Source

Jobe S.O.,Perinatal Research Laboratories | Ramadoss J.,University of Texas Medical Branch | Wargin A.J.,Perinatal Research Laboratories | Magness R.R.,Perinatal Research Laboratories | Magness R.R.,University of Wisconsin - Madison
Hypertension | Year: 2013

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 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) 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-17β, 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-17β and its metabolites stimulated higher prostacyclin/thromboxane ratios in UAECs from the pregnant state compared with UAECs from the nonpregnant state. Estradiol-17β-induced prostacyclin increases were abrogated by the antagonists SC-560 (cyclooxygenase-1), U-51605 (Prostacyclin synthase), ICI 182 780 (ICI; both ER-α/β), and 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4- (2-piperidinyleth oxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-α), but not by 4-[2-phenyl-5,7-bis (trifluoromethyl) pyrazolo[1,5-a]pyrim idin-3-yl]phenol (PHTPP; ER-β). 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-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 ERs. © 2012 American Heart Association, Inc. Source

Jobe S.O.,Perinatal Research Laboratories | Ramadoss J.,Perinatal Research Laboratories | Koch J.M.,Perinatal Research Laboratories | Jiang Y.,Perinatal Research Laboratories | And 3 more authors.
Hypertension | Year: 2010

Estradiol-17β (E2β) and its metabolites, which are sequentially synthesized by cytochrome P450s and catechol-O-methyltransferase to form 2 and 4-hydroxyestradiol (OHE2) and 2-and 4-methoxestradiol (ME2), are elevated during pregnancy. We investigated whether cytochrome P450s and catechol-O- methyltransferase are expressed in uterine artery endothelial cells (UAECs) and whether E2β and its metabolites modulate cell proliferation via ER-α and/or ER-β 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 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 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinyleth oxy)phenol]-1H-pyrazole dihydrochloride, ER-β-specific 4-[2-phenyl-5,7- bis(trifluoromethyl)pyrazolo [1,5-a]pyrim idin-3-yl]phenol antagonists and their respective agonists ER-α-specific 4,4′,4"-(4-propyl-[1H]- pyrazole-1,3,5-triyl)trisphenol and ER-β-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. E2β, 2-OHE2, 4-OHE2, and 4-ME2 treatments concentration-dependently stimulated proliferation in pregnant UAECs but not in nonpregnant UAECs; 2-ME2 did not stimulate proliferation in either cell type. Proliferative responses of pregnant 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 cytochrome P450-and catechol-O-methyltransferase-derived metabolites, and ER-β in uterine angiogenesis regulation during pregnancy that may be dysfunctional in preeclampsia and other cardiovascular disorders. © 2010 American Heart Association, Inc. Source

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