Increased risk and severity of idiopathic pulmonary arterial hypertension (iPAH) is associated with elevated estradiol in men and postmenopausal women. Pulmonary arteries synthesise estradiol via aromatase and metabolise it via CYP1B1 to mitogenic metabolites; SNPs in aromatase and CYP1B1 have been associated with PAH. This suggests that estradiol metabolism could be altered in iPAH. This proof-of-concept study profiles estradiol and several metabolites of estradiol simultaneously in serum from iPAH patients and controls. We show that the estradiol and metabolite profile is altered in iPAH and that 16-hydroxyestrone and 16-hydroxyestradiol accumulate in iPAH patients with 16-hydroxyestrone levels relating to disease severity

Andrew, Ruth

Austin, Eric D

Denver, Nina

Harvey, Katie Y

Homer, Natalie Z M

MacLean, Margaret R

Morrell, Nicholas

English

Homer, Natalie Z

Harvey, Katie

Austin, Eric

Maclean, Margaret

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      EXPRESS: Estrogen metabolites in a small cohort of patientswith idiopathic pulmonary arterial hypertensionCitation for published version:Denver, N, Homer, NZ, Andrew, R, Harvey, K, Morrell, N, Austin, E & Maclean, M 2020, 'EXPRESS:Estrogen metabolites in a small cohort of patients with idiopathic pulmonary arterial hypertension',Pulmonary circulation, pp. 204589402090878. https://doi.org/10.1177/2045894020908783Digital Object Identifier (DOI):10.1177/2045894020908783Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Peer reviewed versionPublished In:Pulmonary circulationGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 22. Dec. 2021Estrogen metabolites in a small cohort of patients with idiopathic pulmonary arterial hypertension Nina Denver MSc1,2, Natalie Homer PhD2, Ruth Andrew PhD2,3, Katie Y Harvey PhD1, Nicholas Morrell PhD6, Eric D. Austin MD MSc4 and Margaret R MacLean PhD1,5 Affiliations 1 Institute of Cardiovascular and Medical Sciences, University of Glasgow, G12 8QQ 2 Mass Spectrometry Core, Edinburgh Clinical Research Facility, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, United Kingdom 3 University/BHF Centre for Cardiovascular Science, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, United Kingdom 4 Vanderbilt University Medical Center, Department of Pediatrics, Medical Center North, Room DD-2211, Nashville, Tennessee, United States 37232 5 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, United Kingdom 6 University of Cambridge, Department of Medicine, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 OQQ  Corresponding author: Margaret R MacLean Strathclyde Institute of Pharmacy and Biomedical Sciences University of Strathclyde HW406 The Hamnett Wing 161, Cathedral Street Glasgow G4 0RE Scotland +44 (0) 141 548 42860 mandy.maclean@strath.ac.uk     Increased risk and severity of idiopathic pulmonary arterial hypertension (iPAH) is associated with elevated plasma estradiol (E2) in both men and postmenopausal women (1, 2), suggesting that E2 is not ovarian-derived. Aromatase is responsible for the synthesis of E2. Remodelled pulmonary arteries, lesions and human pulmonary artery smooth muscle cells (hPASMCs) from PAH patients have high levels of aromatase (3). CYP1B1 can convert E2 to mitogenic 16-hydroxy-estrogens (16OHEs) and this enzyme is over-expressed in diseased pulmonary arteries from PAH patients (4). Existing assays have not been sensitive or specific enough to detect levels of many E2 metabolites in PAH patient blood. Using a newly developed liquid chromatography tandem mass spectrometry (LC-MS/MS) assay (5), here we investigate the profile of several E2 metabolites in iPAH patients. Methods Serum from 22 subjects with iPAH (12 males, 10 females) and 34 healthy control subjects (17 males, 17 females) were analysed by LC-MS/MS for estrone (E1), 17α-estradiol (17αE2), 17β-estradiol (E2), 16-Hydroxyestrone (16OHE1), 16-Hydroxyestradiol (16OHE2), 2-methoxyestrone (2MeOE1), 4-methoxyestrone (4MeOE1), 2-methoxyestradiol (2MeOE2) and 4-methoxyestradiol (4MeOE2) (5). Data were compared by a Mann-Whitney U-test with associations assessed using Pearson’s correlations. Values below the confirmed limit of quantification (LOQ) were imputed as the LOQ (2pg/mL E1, E2, 16OHE2, 2MeOE1 and 6pg/mL 16OHE1).  To determine if 16OHE2 was present at bioactive concentrations, proliferation was studied in hPASMCs and cell migration studied in human blood outgrowth endothelial cells (BOECs). hPASMCs were isolated from distal pulmonary arteries (0.3 – 1 mm diameter) from macroscopically normal controls or patients undergoing lung transplantation. BOECs were isolated from PAH patients as described previously (6) with cells cultured as described previously (7). The patients were male and female (heritable PAH/iPAH).  Cells were stimulated with 16OHE2 following preliminary concentration-response analysis (10nM hPASMCs and 1nM BOECs) for 48 hours in phenol red-free media.  Cell counts and cell migration (Corning® Transwell inserts) were assessed using a Countess II FL cell counter (Life Technologies, UK). A student’s t-test was applied for comparison of 16OHE2 vs vehicle treated cells for each group. Results E1 was decreased in serum of female iPAH patients whilst 16OHE2 was increased. E1, E2 and 16OHE1 were elevated in male iPAH patients (Table 1). Comparing males and females, E1 was higher in female controls compared with male controls (p=0.01). In iPAH, E1 was higher in males than females (P=0.03).  In female iPAH patients, increased E1 was associated with increasing PAWP (R2= 0.5, β= 0.7, p=0.03), decreased E2 was associated with increasing age (R2=0.7, β= -0.8, p=0.02), and increasing E2 with increasing PAWP (R2=0.6, β = 0.6, p= 0.05) while increased 16OHE1 correlated with a reduction of cardiac output (R2=0.7, β = -0.6, p=0.01). In male iPAH, increased E2 was associated with increased PAWP (R2=0.5, β=0.6, p=0.02) while increased E1 and E2 associated with increasing BNP levels (R2=0.6, β=0.8, p=0.01 and R2=0.4, β=0.6, p=0.04). 16OHE2 caused proliferation of hPASMCs from female PAH patients (by 63%) but not controls (male or female) (Figure 1A-C). 16OHE2 caused increased migration of BOECs from PAH patients, both in males (by 55%) and females (by 67%) (Figure 1D).  Discussion This study is the first to simultaneously quantify several estrogens in controls and PAH patients as our methodological paper measured levels in pooled plasma samples (5). LC-MS/MS allowed discrimination between isomers of estradiol and its bioactive metabolites such as 16OHE1, 16OHE2 and methoxyestrogens. Increased 16-hydroxylation of E1/E2 promotes formation of proliferative metabolites and we identified accumulation of 16OHE2 in female iPAH patient serum for the first time. We confirmed increased levels of 16OHE1 in patients which had been suggested by previous studies (8, 9). In female iPAH, 16OHE2 was also increased. There was increased E1 and E2 levels in male iPAH patients, consistent with a previous study using immunoassay detection (1). The E2 concentrations we determined here were lower compared with previous studies determined using immunoassays.  This is consistent with immunoassays being limited by cross-reactivity and over-estimation of E2 levels compared with LC-MS/MS (10).   Aromatase and CYP1B1 are increased in PAH patient pulmonary arteries and pulmonary vascular lesions (3, 4). Increased aromatase activity may account for increased plasma E2 in men and post-menopausal women with iPAH (1, 2). In female iPAH, there was a decrease in E1 with no change in E2. The change in E1 may reflect an increased conversion to 16OHEs via CYP enzymes as reflected by increased 16OHE1 and 16OHE2 in these patients. Where there is a decrease in E1 with no change in E2 this could also be due to the increased metabolism of E2. Baird et al. recently demonstrated that E2 levels are elevated in post-menopausal iPAH female patients compared with controls; however, E2 levels from controls in that study were 5-7 pg/mL, commensurate with post-menopause levels (2). The majority of females in this current study were pre-menopausal, with sampling not routinely timed to consider menstrual cycle, and mean E2 concentrations in controls and patients were ~34 pg/mL. Hence, it is perhaps unlikely that a further increase would be expected in this patient cohort. Future clinical studies could consider blood sampling from pre-menopausal women at the same stage of the menstrual cycle. 3  As 16OHE1 can be derived directly from E1 and male patients have higher E1, this may also contribute to the elevated 16OHE1 in these patients. Consistent with increased 16OHEs observed in iPAH patients, polymorphisms in CYP1B1 are observed in PAH patients associated with functional differences in 16-hydroxylation, cancer risk and E2-mediated carcinogenicity (11, 12). Conversion of E2 to 16OHE1 by CYP1B1 may contribute to the pathogenesis of experimental pulmonary hypertension and cause proliferation of hPASMCs via increased oxidative stress (7, 8).  We have already demonstrated that 16OHE1 can cause proliferation of both male and female hPASMCs and male mouse PASMCs at concentrations observed in plasma (4, 7, 13). 16OHE1 acts through the ERa receptor in hPASMCs (7, 14) and indeed we have shown that ERa receptors are more highly expressed in female PAH hPASMCs (15). This is consistent with female hPASMCs being more proliferative to E2, also due to decreased basal bone morphogenetic protein receptor II (BMPRII) signalling in female hPASMCs [16]. 16OHE2 is a novel metabolite with unknown effects that was increased in the serum from female iPAH patients compared with controls. This could also be due to altered CYP1B1-mediated 16-hydroxlation (4, 13). 16OHE2 is an E2-receptor agonist, capable of cellular proliferation associated with cancer risk and E2-mediated carcinogenicity (12). Consistent with this, we now show that, at a pathophysiological concentration, 16OHE2 can also induce proliferation in female human PAH PASMCs with no effect in female or male control PASMCs where ERa expression is not so high. However, we show that 16OHE2 increases cell migration in both male and female PAH BOECs. As 16OHE1 has been shown to act as a ‘second hit’ to uncover a pulmonary hypertensive phenotype in BMPRII mutant male and female mice [9], this suggests that both 16OHE1 and 16OHE2 may also facilitate pulmonary vascular remodelling in males and females by acting as a ‘second hit’ to BMPRII mutations. 16OHE2 is also produced in abundance during pregnancy and it is interesting to speculate that it may contribute to pregnancy being a risk factor for PAH (17).  It should be noted that all of the medications that the patients were prescribed are metabolised in the liver by CYP enzymes such as CYP3A4 and CYP2C9/8 and have no known effects on lung or liver CYP1B1 or CYP1A1 activity. One limitation of this study is that the patients were older than the controls. However E2 and 16OHE1 levels correlated with indicators of disease severity within the patient groups and decreased E2 was associated with increasing age in iPAH patients. This suggests that increased E2/metabolite levels in PAH patients and are related to disease status rather than difference in age. The results suggest that estrogen metabolism is altered in PAH and both 16OHE1 and 16OHE2 accumulate, with 16OHE1 levels relating to disease severity. This study is limited by the small size but important as it warrants further multi-center fully powered studies to confirm and correlate metabolite levels with severity and sub-groups of PAH. Future studies on CYP enzymes and CYP1B1 inhibitors in patients with PAH may also be valuable.   References 1. Ventetuolo CE, Baird GL, Barr RG, et al. Higher Estradiol and Lower Dehydroepiandrosterone-Sulfate Levels Are Associated with Pulmonary Arterial Hypertension in Men. Am J Respir Crit Care Med 2016; 193: 1168-1175. 2. Baird GL, Archer-Chicko C, Barr RG, et al. Lower DHEA-S levels predict disease and worse outcomes in post-menopausal women with idiopathic, connective tissue disease- and congenital heart disease-associated pulmonary arterial hypertension. Eur Respir J 2018; 51. 3. Mair KM, Wright AF, Duggan N, et al. Sex-dependent influence of endogenous estrogen in pulmonary hypertension. Am J Respir Crit Care Med 2014; 190: 456-467. 4. White K, Johansen AK, Nilsen M, et al. Activity of the Estrogen-Metabolizing Enzyme Cytochrome P450 1B1 Influences the Development of Pulmonary Arterial Hypertension / Clinical Perspective. Circulation 2012; 126: 1087-1098. 5. Denver N, Khan S, Stasinopoulos I, et al. Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry. Analytica chimica acta 2019; 1054: 84-94. 6. Ormiston ML, Toshner MR, Kiskin FN, et al. Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood. J Vis Exp. 2015;(106):e53384. 7. Hood KY, Montezano AC, Harvey AP, et al. Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Mediated Redox Signaling and Vascular Remodeling by 16alpha-Hydroxyestrone in Human Pulmonary Artery Cells: Implications in Pulmonary Arterial Hypertension. Hypertension 2016; 68: 796-808. 8. Austin ED, Cogan JD, West JD, et al. Alterations in oestrogen metabolism: implications for higher penetrance of familial pulmonary arterial hypertension in females. Eur Respir J. 2009; 34:1093-1099. 9. Chen X, Talati M, Fessel JP, et al. Estrogen Metabolite 16α-Hydroxyestrone Exacerbates Bone Morphogenetic Protein Receptor Type II-Associated Pulmonary Arterial Hypertension Through MicroRNA-29-Mediated Modulation of Cellular Metabolism. Circulation 2016; 133:82-97. 10. Jessica M. Faupel-Badger, Barbara J. Fuhrman, et al. Comparison of liquid chromatography-mass spectrometry, radioimmunoassay, and enzyme-linked immunosorbent assay methods for measurement of urinary estrogens, Cancer Epidemiol Biomarkers Prev 2010; 19: 292–300. 11. Ventetuolo CE, Mitra N, Wan F et al.  Oestradiol metabolism and androgen receptor genotypes are associated with right ventricular function. Eur Respir J 2016; 47: 553-563. 12. Gupta M, McDougal A, Safe S. Estrogenic and antiestrogenic activities of 16alpha- and2-hydroxy metabolites of 17beta-estradiol in MCF-7 and T47D human breast cancer cells. J Steroid Biochem Mol Biol. 1998; 67:413-419. 13. Mair KM, Harvey KY, Henry AD, et al. Obesity Alters Oestrogen Metabolism and Contributes to Pulmonary Arterial Hypertension. European Respiratory Journal 2019; 1801524. 14. Johansen AK, Dean A, Morecroft I, et al. The serotonin transporter promotes a pathological estrogen metabolic pathway in pulmonary hypertension via cytochrome P450 1B1. Pulmonary Circulation 2016; 6:82-92. 15. Wright AF, Ewart MA, Mair K, et al. Oestrogen receptor alpha in pulmonary hypertension. Cardiovasc Res. 2015; 106:206-216. 16. Mair KM, Yang XD, Long L, et al. Sex affects bone morphogenetic protein type II receptor signalling in pulmonary artery smooth muscle cells. Am J Respir Crit Care Med. 2015; 191(6):693-703. 17. Hemnes AR, Kiely DG, Cockrill BA, et al. Statement on pregnancy in pulmonary hypertension from the Pulmonary Vascular Research Institute. Pulmonary Circulation 2015; 5: 435-465. Table 1: Clinical Characteristics and estrogen Concentrations for Controls vs iPAH Patients  Female Controls Female iPAH P-Values Male  Controls Male iPAH P-Values Subjects 17 10  17 12  Clinical  Characteristics       Age (y)  39.0 [31 – 45] 56.5 [43 - 61] 0.01 32.5 [27 – 37] 57.0 [50 – 62] 0.001 BMI (kg·m−2) 24.9 [22 – 28]  29.1 [28 – 31] 0.06 23.8 [22 – 33] 32.8 [29 – 34] 0.07 Right Atrial Pressure (mmHg)  6.0 [4 – 10]   9.0 [7 - 14] 0.23 mPAP (mmHg)  55.0 [35 - 62]   51.5 [40 - 60] 0.97 Cardiac Output (L/min)  5.3 [5 - 7]   4.6 [4 - 6] 0.38 PAWP (mmHg)  9.0 [8 - 12]   11.0 [9 - 16] 0.23 PVR (mmHg/l∙min)  9.0 [4 - 13]   9.1 [4 -11] 0.97 BNP (pg/mL)  76.0 [25 - 238]   114.0 [21 - 350] 0.79 Current PAH Medications       ERA  3   4  PDE5 Inhibitor  2   4  Epoprostenol or Trepostinil  2   2  Calcium Channel Blocker  -   1  Treatment Naïve  -   1  Estrogen Concentrations (pg/mL)       E1 34.9 [27 – 75] (100) 16.9 [9 – 34] (91) 0.02 23.6 [17 – 32] (100) 28.1 [24 – 55] (100) 0.05 E2 16.8 [10 – 59] (100) 7.9 [4 – 65] (100) 0.15 16.0 [10 – 21] (100) 21.3 [19 – 30] (100) 0.02 16OHE1 24.8 [11 – 38] (65) 29.8 [17 – 53] (91) 0.09 24.1 [19 – 32] (47) 79.9 [58 - 96] (75) 0.01 16OHE2 10.7 [9 – 11] (18) 14.6 [8 – 26] (82) 0.01 15.4 [8 – 18] (41) 15.9 [6 – 3]  (67) 0.24 2MeOE1 8.4 [6 – 26] (41) 6.9 [6 – 80] (55) 1.00 10.9 [9 – 26] (30) 13.2 (8) - 4MeOE1 ND ND - ND ND - 2MeOE2 ND ND - ND ND - 4MeOE2 ND ND - ND ND - <50 y Estrogen Concentrations (pg/mL) n=14 n=4     E1 33.7 [27 - 48] 39.5 [24 - 52] 1.00    E2 24.0 [12 - 64] 47.7 [25 - 88] 0.57    16OHE1 21.9 [12 - 35] 34.4 [16 - 52] 0.23    16OHE2 10.1 [9 - 11] 14.0 [10 - 15] 0.06    2MeOE1 8.4 [7 - 18] 7.0 0.64    ≥ 50 y Estrogen Concentrations (pg/mL) n=3 n=6     E1 78.6 [56 - 107] 12.1 [9 - 19] 0.02    E2 9.6 [7 -13] 4.6 [3 - 7] 0.26    16OHE1 37.7 29.8 [27 - 38] 0.38    16OHE2 16.5 21.1 [9 - 28] 0.26    2MeOE1 16.0 [11 – 21] 6.5 [6 - 36] 0.90    Endothelin receptor antagonist, ERA; Phosphodiesterase type 5 inhibitor, PDE5; Pulmonary arterial hypertension, PAH; idiopathic PAH, iPAH; Mean pulmonary artery pressure, mPAP; Pulmonary artery wedge pressure, PAWP; Pulmonary Vascular Resistance, PVR; B-type natriuretic peptide, BNP; Not Detected, ND; Estrone, E1; Estradiol, E2; 16-Hydroxyestrone, 16OHE1; 16-Hydroxyestradiol, 16OHE2; 2-Methoxyestrone, 2MeOE1; 4-Methoxyestrone, 4MeOE1; 2-Methoxyestradiol, 2MeOE2; 4-Methoxyestradiol, 4MeOE2. Medication details of 3 female patients is unknown. Clinical characteristics and estrogen Levels shown as median [Q1 – Q3] (% LC-MS/MS Detection). P-values following a Mann Whitney U test in control vs iPAH or female iPAH vs male iPAH clinical characteristics.        Figure 1: 16OHE2 stimulation increases proliferation and migration of human cells  10 nM 16OHE2 increases proliferation in female PAH (A) with no effect on female control (B) and male control (C) human PASMCs. 1 nM 16OHE2 increased migration of male and female BOECs derived from PAH patients (D). Data shown as Mean ± SEM (n= 4 - 6) with statistics performed by comparison of 16OHE2 to veh by a student’s t test *p<0.05, **p<0.01. Veh = Vehicle Control, Ethanol. 

EXPRESS: Estrogen metabolites in a small cohort of patients with idiopathic pulmonary arterial hypertension

Homer, Natalie Z.M.

Harvey, Katie Y.

Austin, Eric D.

Maclean, Margaret R.

Enlighten: Publications

Estrogen metabolites in a small cohort of patients with idiopathic pulmonary arterial hypertension

Apollo (Cambridge)

Research LetterEstrogen metabolites in a small cohort of patients withidiopathic pulmonary arterial hypertensionNina Denver1,2, Natalie Z.M. Homer2, Ruth Andrew2,3, Katie Y. Harvey1, Nicholas Morrell4,Eric D. Austin5 and Margaret R. MacLean1,61Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK; 2Mass Spectrometry Core, Edinburgh Clinical Research Facility, Queen’sMedical Research Institute, Edinburgh, UK; 3University/BHF Centre for Cardiovascular Science, Queen’s Medical Research Institute, Edinburgh, UK; 4Departmentof Medicine, University of Cambridge, Cambridge, UK; 5Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA; 6Strathclyde Instituteof Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UKAbstractIncreased risk and severity of idiopathic pulmonary arterial hypertension (iPAH) is associated with elevated estradiol in men andpostmenopausal women. Pulmonary arteries synthesise estradiol via aromatase and metabolise it via CYP1B1 to mitogenicmetabolites; SNPs in aromatase and CYP1B1 have been associated with PAH. This suggests that estradiol metabolism could bealtered in iPAH. This proof-of-concept study profiles estradiol and several metabolites of estradiol simultaneously in serum fromiPAH patients and controls. We show that the estradiol and metabolite profile is altered in iPAH and that 16-hydroxyestrone and16-hydroxyestradiol accumulate in iPAH patients with 16-hydroxyestrone levels relating to disease severity.Keywordspulmonary hypertension experimental, metabolism, sex, pulmonary hypertensionDate received: 28 October 2019; accepted: 1 February 2020Pulmonary Circulation 2020; 10(1) 1–5DOI: 10.1177/2045894020908783IntroductionIncreased risk and severity of idiopathic pulmonary arterialhypertension (iPAH) is associated with elevated plasmaestradiol in both men and postmenopausal women,1,2 sug-gesting that estradiol is not ovarian-derived. Aromatase isresponsible for the synthesis of estradiol. Remodeled pul-monary arteries, lesions, and human pulmonary arterysmooth muscle cells (hPASMCs) from pulmonary arterialhypertension patients have high levels of aromatase.3Cytochrome P450 Family 1 Subfamily B Member 1(CYP1B1) can convert estradiol to mitogenic 16-hydroxyes-trogens and this enzyme is over-expressed in diseased pul-monary arteries from pulmonary arterial hypertension(PAH) patients.4 Existing assays have not been sensitive orspecific enough to detect levels of many estradiol metabol-ites in PAH patient blood. Using a newly developed liquidchromatography tandem mass spectrometry (LC-MS/MS)assay,5 here we investigate the profile of several estradiolmetabolites in iPAH patients.MethodsSerum from 22 subjects with iPAH (12 males, 10 females)and 34 healthy control subjects (17 males, 17 females)were analyzed by LC-MS/MS for estrone (E1), 17a-estradiol (17aE2), 17-estradiol (E2), 16-hydroxyestrone(16OHE1), 16-hydroxyestradiol (16OHE2), 2-methoxyes-trone (2MeOE1), 4-methoxyestrone (4MeOE1), 2-methox-yestradiol (2MeOE2), and 4-methoxyestradiol (4MeOE2).5Data were compared by a Mann–Whitney U test with asso-ciations assessed using Pearson’s correlations. Values belowthe confirmed limit of quantification (LOQ) were imputed asthe LOQ (2 pg/mL E1, E2, 16OHE2, 2MeOE1, and 6 pg/mL16OHE1).Corresponding author:Margaret R. MacLean, Strathclyde Institute of Pharmacy and BiomedicalSciences, University of Strathclyde, HW406, The Hamnett Wing, 161,Cathedral Street, Glasgow G4 0RE, UKEmail: mandy.maclean@strath.ac.ukCreative Commons CC-BY: This article is distributed under the terms of the Creative Commons Attribution 4.0License (http://creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of thework without further permission provided the original work is attributed as specified on the SAGE and Open Access pages(https://us.sagepub.com/en-us/nam/open-access-at-sage).! The Author(s) 2020.journals.sagepub.com/home/pulTo determine if 16OHE2 was present at bioactiveconcentrations, proliferation was studied in hPASMCs andcell migration was studied in human blood outgrowthendothelial cells (BOECs). hPASMCs were isolated fromdistal pulmonary arteries (0.3–1mm diameter) frommacroscopically normal controls or patients undergoinglung transplantation. BOECs were isolated from PAHpatients as described previously6 with cells cultured asdescribedpreviously.7Thepatientsweremale and female (her-itable PAH/iPAH). Cells were stimulated with 16OHE2 fol-lowing preliminary concentration-response analysis (10 nMhPASMCs and 1 nM BOECs) for 48 h in phenol red-freemedia. Cell counts and cell migration (Corning Transwellinserts) were assessed using a Countess II FL cell counter(Life Technologies, UK). A student’s t-test was applied forcomparison of 16OHE2 vs vehicle-treated cells for eachgroup.ResultsE1 was decreased in serum of female iPAH patients whilst16OHE2 was increased. E1, E2, and 16OHE1 were elevatedin male iPAH patients (Table 1). Comparing males andfemales, E1 was higher in female controls compared withmale controls (p¼ 0.01). In iPAH, E1 was higher in malesthan females (p¼ 0.03).In female iPAH patients, increased E1 was associatedwith increasing pulmonary artery wedge pressure (PAWP)(R2¼ 0.5, ¼ 0.7, p¼ 0.03), decreased E2 was associatedwith increasing age (R2¼ 0.7, ¼ –0.8, p¼ 0.02), andincreasing E2 with increasing PAWP (R2¼ 0.6, ¼ 0.6,p¼ 0.05) while increased 16OHE1 correlated with a reduc-tion of cardiac output (R2¼ 0.7, ¼ –0.6, p¼ 0.01). In maleiPAH, increased E2 was associated with increased PAWP(R2¼ 0.5, ¼ 0.6, p¼ 0.02) while increased E1 and E2 asso-ciated with increasing BNP levels (R2¼ 0.6, ¼ 0.8, p¼ 0.01and R2¼ 0.4, ¼ 0.6, p¼ 0.04).16OHE2 caused proliferation of hPASMCs from femalePAH patients (by 63%) but not controls (male or female)(Fig. 1a–c). 16OHE2 caused increased migration of BOECsfrom PAH patients, both in males (by 55%) and females(by 67%) (Fig. 1d).DiscussionThis study is the first to simultaneously quantify severalestrogens in controls and PAH patients as our methodo-logical paper measured levels in pooled plasma samples.5LC-MS/MS allowed discrimination between isomers of E2and its bioactive metabolites such as 16OHE1, 16OHE2,and methoxyestrogens. Increased 16-hydroxylation of E1/E2 promotes formation of proliferative metabolites and weidentified accumulation of 16OHE2 in female iPAH patientserum for the first time. We confirmed increased levels of16OHE1 in patients which had been suggested by previousstudies.8,9 In female iPAH, 16OHE2 was also increased.There was increased E1 and E2 levels in male iPAH patients,consistent with a previous study using immunoassay detec-tion.1 The E2 concentrations we determined here were lowercompared with previous studies determined using immuno-assays. This is consistent with immunoassays being limitedby cross-reactivity and over-estimation of E2 levels com-pared with LC-MS/MS.10Aromatase andCYP1B1 are increased in PAHpatient pul-monary arteries and pulmonary vascular lesions.3,4 Increasedaromatase activity may account for increased plasma E2 inmen and post-menopausal women with iPAH.1,2 In femaleiPAH, there was a decrease in E1 with no change in E2. Thechange in E1 may reflect an increased conversion to 16OHEsvia CYP enzymes as reflected by increased 16OHE1 and16OHE2 in these patients. Where there is a decrease in E1with no change in E2, this could also be due to the increasedmetabolism of E2. Baird et al. recently demonstrated that E2levels are elevated in post-menopausal iPAH female patientscompared with controls; however, E2 levels from controls inthat study were 5–7 pg/mL, commensurate with post-meno-pause levels.2 The majority of females in this current studywere pre-menopausal, with sampling not routinely timed toconsidermenstrual cycle, andmeanE2 concentrations in con-trols and patients were 34 pg/mL. Hence, it is perhaps unli-kely that a further increase would be expected in this patientcohort. Future clinical studies could consider blood samplingfrom pre-menopausal women at the same stage of the men-strual cycle.3As 16OHE1 can be derived directly from E1 andmale patients have higher E1, this may also contribute tothe elevated 16OHE1 in these patients. Consistent withincreased 16OHEs observed in iPAH patients, polymorph-isms in CYP1B1 are observed in PAH patients associatedwith functional differences in 16-hydroxylation, cancerrisk, and E2-mediated carcinogenicity.11,12 Conversionof E2 to 16OHE1 by CYP1B1 may contribute to thepathogenesis of experimental pulmonary hypertensionand cause proliferation of hPASMCs via increased oxidativestress.7,8We have already demonstrated that 16OHE1 can causeproliferation of both male and female hPASMCs and malemouse PASMCs at concentrations observed in plasma.4,7,1316OHE1 acts through the ERa receptor in hPASMCs7,14and indeed we have shown that ERa receptors are morehighly expressed in female PAH hPASMCs.15 This is con-sistent with female hPASMCs being more proliferative toE2, also due to decreased basal bone morphogenetic proteinreceptor II (BMPRII) signaling in female hPASMCs.1616OHE2 is a novel metabolite with unknown effects thatwas increased in the serum from female iPAH patients com-pared with controls. This could also be due to alteredCYP1B1-mediated 16-hydroxlation.4,13 16OHE2 is an E2-receptor agonist, capable of cellular proliferation associatedwith cancer risk and E2-mediated carcinogenicity.12Consistent with this, we now show that, at a pathophysio-logical concentration, 16OHE2 can also induce proliferation2 | Estrogen metabolites in a small cohort of patients with iPAH Denver et al.in female human PAH PASMCs with no effect in female ormale control PASMCs where ERa expression is not so high.However, we show that 16OHE2 increases cell migration inboth male and female PAH BOECs. As 16OHE1 has beenshown to act as a ‘‘second hit’’ to uncover a pulmonaryhypertensive phenotype in BMPRII mutant male andfemale mice,9 this suggests that both 16OHE1 and16OHE2 may also facilitate pulmonary vascular remodelingTable 1. Clinical characteristics and estrogen concentrations for controls vs iPAH patients.Female controls Female iPAH P-Values Male controls Male iPAH P-ValuesSubjects 17 10 17 12Clinical characteristicsAge (y) 39.0 (31–45) 56.5 (43–61) 0.01 32.5 (27–37) 57.0 (50–62) 0.001BMI (kgm–2) 24.9 (22–28) 29.1 (28–31) 0.06 23.8 (22–33) 32.8 (29–34) 0.07Right atrial pressure (mmHg) 6.0 (4–10) 9.0 (7–14) 0.23mPAP (mmHg) 55.0 (35–62) 51.5 (40–60) 0.97Cardiac output (L/min) 5.3 (5–7) 4.6 (4–6) 0.38PAWP (mmHg) 9.0 (8–12) 11.0 (9–16) 0.23PVR (mmHg/lmin) 9.0 (4–13) 9.1 (4–11) 0.97BNP (pg/mL) 76.0 (25–238) 114.0 (21–350) 0.79Current PAH medicationsERA 3 4PDE5 inhibitor 2 4Epoprostenol or treprostinil 2 2Calcium channel blocker – 1Treatment naı¨ve – 1Estrogen concentrations (pg/mL)E1 34.9 (27–75) (100) 16.9 (9–34) (91) 0.02 23.6 (17–32) (100) 28.1 (24–55) (100) 0.05E2 16.8 (10–59) (100) 7.9 (4–65) (100) 0.15 16.0 (10–21) (100) 21.3 (19–30) (100) 0.0216OHE1 24.8 (11–38) (65) 29.8 (17–53) (91) 0.09 24.1 (19–32) (47) 79.9 (58–96) (75) 0.0116OHE2 10.7 (9–11) (18) 14.6 (8–26) (82) 0.01 15.4 (8–18) (41) 15.9 (6–33) (67) 0.242MeOE1 8.4 (6–26) (41) 6.9 (6–80) (55) 1.00 10.9 (9–26) (30) 13.2 (8) –4MeOE1 ND ND – ND ND –2MeOE2 ND ND – ND ND –4MeOE2 ND ND – ND ND –<50 y estrogenconcentrations (pg/mL)n¼ 14 n¼ 4E1 33.7 (27–48) 39.5 (24–52) 1.00E2 24.0 (12–64) 47.7 (25–88) 0.5716OHE1 21.9 (12–35) 34.4 (16–52) 0.2316OHE2 10.1 (9–11) 14.0 (10–15) 0.062MeOE1 8.4 (7–18) 7.0 0.6450 y estrogenconcentrations (pg/mL)n¼ 3 n¼ 6E1 78.6 (56–107) 12.1 (9–19) 0.02E2 9.6 (7–13) 4.6 (3–7) 0.2616OHE1 37.7 29.8 (27–38) 0.3816OHE2 16.5 21.1 (9–28) 0.262MeOE1 16.0 (11–21) 6.5 (6–36) 0.90BMI: body mass index; ERA: endothelin receptor antagonist; PDE5: phosphodiesterase type 5 inhibitor; PAH: pulmonary arterial hypertension; iPAH: idiopathicPAH; mPAP: mean pulmonary artery pressure; PAWP: pulmonary artery wedge pressure; PVR: pulmonary vascular resistance; BNP: B-type natriureticpeptide; ND: not detected; E1: estrone; E2: 17-estradiol; 16OHE1: 16-hydroxyestrone; 16OHE2: 16-hydroxyestradiol; 2MeOE1: 2-methoxyestrone; 4MeOE1:4-methoxyestrone; 2MeOE2: 2-methoxyestradiol; 4MeOE2: 4-methoxyestradiol.Notes: Medication details of three female patients is unknown. Clinical characteristics and estrogen levels shown as median (Q1–Q3) (% LC-MS/MS detection).P-values following a Mann–Whitney U test in control vs iPAH or female iPAH vs male iPAH clinical characteristics.Pulmonary Circulation Volume 10 Number 1 | 3in males and females by acting as a ‘‘second hit’’ to BMPRIImutations. 16OHE2 is also produced in abundance duringpregnancy and it is interesting to speculate that it may con-tribute to pregnancy being a risk factor for PAH.17It should be noted that all of the medications that thepatients were prescribed are metabolized in the liver by CYPenzymes such as CYP3A4 and CYP2C9/8 and have noknown effects on lung or liver CYP1B1 or CYP1A1 activity.One limitation of this study is that the patients were olderthan the controls. However E2 and 16OHE1 levels corre-lated with indicators of disease severity within the patientgroups and decreased E2 was associated with increasing agein iPAH patients. This suggests that increased E2/metabolitelevels in PAH patients are related to disease status ratherthan difference in age.The results suggest that estrogen metabolism isaltered in PAH and both 16OHE1 and 16OHE2 accumu-late, with 16OHE1 levels relating to disease severity.This study is limited by the small size but important as itwarrants further multi-center fully powered studies toconfirm and correlate metabolite levels with severity andsub-groups of PAH. Future studies on CYP enzymes andCYP1B1 inhibitors in patients with PAH may also bevaluable.Ethical approvalYes.GuarantorMargaret R. MacLean.Author contributionsNina Denver: all LC-MS/MS, data analysis, hPASMC experi-ments, and preparation of manuscript; Natalie Homer and RuthAndrew: analysis of LC-MS/MS; Katie Y. Harvey: BOEC experi-ments; Nicholas Morrell: isolation of BOECs; Eric D. Austin:blood sampling from all patients and controls; and Margaret R.MacLean: PI, grant holder, preparation of manuscript, and dataanalysis.Conflict of interestThe author(s) declare that there is no conflict of interest.FundingThe project described was supported by the British HeartFoundation, through grants RE/13/5/30177 and RG/16/2/32153,the Wellcome Trust (108468/z/15/z), the National Institutes ofHealth (R01HL134802 and P01HL108800), and a BBSRCiCASE PhD award (BB/N503691/1).Fig. 1. 16OHE2 stimulation increases proliferation and migration of human cells. 10 nM 16OHE2 increases proliferation in female PAH (a) withno effect on female control (b) and male control (c) human PASMCs. 1 nM 16OHE2 increased migration of male and female BOECs derived fromPAH patients (d).Veh: Vehicle control, Ethanol; 16OHE2: 16-hydroxyestradiol; PAH-BOECs: Blood outgrowth endothelial cell from pulmonary arterial hyperten-sion patients.Note: Data shown as mean SEM (n¼ 4–6) with statistics performed by comparison of 16OHE2 to veh by a Student’s t test *p< 0.05.4 | Estrogen metabolites in a small cohort of patients with iPAH Denver et al.References1. Ventetuolo CE, Baird GL, Barr RG, et al. Higher estradiol andlower dehydroepiandrosterone-sulfate levels are associated withpulmonary arterial hypertension in men. Am J Respir Crit CareMed 2016; 193: 1168–1175.2. Baird GL, Archer-Chicko C, Barr RG, et al. Lower DHEA-Slevels predict disease and worse outcomes in post-menopausalwomen with idiopathic, connective tissue disease- and congeni-tal heart disease-associated pulmonary arterial hypertension.Eur Respir J 2018; 51: pii: 1800467.3. Mair KM, Wright AF, Duggan N, et al. Sex-dependent influ-ence of endogenous estrogen in pulmonary hypertension. Am JRespir Crit Care Med 2014; 190: 456–467.4. White K, Johansen AK, Nilsen M, et al. Activity of the estro-gen-metabolizing enzyme cytochrome P450 1B1 influences thedevelopment of pulmonary arterial hypertension/clinical per-spective. Circulation 2012; 126: 1087–1098.5. Denver N, Khan S, Stasinopoulos I, et al. Derivatizationenhances analysis of estrogens and their bioactive metabolitesin human plasma by liquid chromatography tandem mass spec-trometry. Analytica Chimica Acta 2019; 1054: 84–94.6. Ormiston ML, Toshner MR, Kiskin FN, et al. Generation andculture of blood outgrowth endothelial cells from human per-ipheral blood. J Vis Exp 2015; 106: e53384.7. Hood KY, Montezano AC, Harvey AP, et al. Nicotinamideadenine dinucleotide phosphate oxidase-mediated redox signal-ing and vascular remodeling by 16alpha-hydroxyestrone inhuman pulmonary artery cells: implications in pulmonary arter-ial hypertension. Hypertension 2016; 68: 796–808.8. Austin ED, Cogan JD, West JD, et al. Alterations in oestrogenmetabolism: implications for higher penetrance of familial pul-monary arterial hypertension in females. Eur Respir J 2009; 34:1093–1099.9. Chen X, Talati M, Fessel JP, et al. Estrogen metabolite16a-hydroxyestrone exacerbates bone morphogenetic proteinreceptor type II-associated pulmonary arterial hypertensionthrough microRNA-29-mediated modulation of cellularmetabolism. Circulation 2016; 133: 82–97.10. Faupel-Badger JM, Fuhrman BJ, Xu X, et al. Comparison ofliquid chromatography-mass spectrometry, radioimmuno-assay, and enzyme-linked immunosorbent assay methods formeasurement of urinary estrogens. Cancer EpidemiolBiomarkers Prev 2010; 19: 292–300.11. Ventetuolo CE, Mitra N, Wan F, et al. Oestradiol metabolismand androgen receptor genotypes are associated with right ven-tricular function. Eur Respir J 2016; 47: 553–563.12. Gupta M, McDougal A and Safe S. Estrogenic and antiestro-genic activities of 16alpha- and2-hydroxy metabolites of17beta-estradiol in MCF-7 and T47D human breast cancercells. J Steroid Biochem Mol Biol 1998; 67: 413–419.13. Mair KM, Harvey KY, Henry AD, et al. Obesity alters oes-trogen metabolism and contributes to pulmonary arterialhypertension. Eur Respir J 2019; 53: 1801524.14. Johansen AK, Dean A, Morecroft I, et al. The serotonin trans-porter promotes a pathological estrogen metabolic pathway inpulmonary hypertension via cytochrome P450 1B1. Pulm Circ2016; 6: 82–92.15. Wright AF, Ewart MA, Mair K, et al. Oestrogen receptoralpha in pulmonary hypertension. Cardiovasc Res 2015; 106:206–216.16. Mair KM, Yang XD, Long L, et al. Sex affects bone morpho-genetic protein type II receptor signalling in pulmonary arterysmooth muscle cells. Am J Respir Crit Care Med 2015; 191:693–703.17. Hemnes AR, Kiely DG, Cockrill BA, et al. Statement onpregnancy in pulmonary hypertension from the pulmonaryvascular research institute. Pulm Circ 2015; 5: 435–465.Pulmonary Circulation Volume 10 Number 1 | 5

Estrogen metabolites in a small cohort of patients with idiopathic pulmonary arterial hypertension.

Homer, Natalie ZM

MacLean, Margaret R.

Research LetterEstrogen metabolites in a small cohort of patients withidiopathic pulmonary arterial hypertensionNina Denver1,2, Natalie Z.M. Homer2, Ruth Andrew2,3, Katie Y. Harvey1, Nicholas Morrell4,Eric D. Austin5 and Margaret R. MacLean1,61Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK; 2Mass Spectrometry Core, Edinburgh Clinical Research Facility, Queen’sMedical Research Institute, Edinburgh, UK; 3University/BHF Centre for Cardiovascular Science, Queen’s Medical Research Institute, Edinburgh, UK; 4Departmentof Medicine, University of Cambridge, Cambridge, UK; 5Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA; 6Strathclyde Instituteof Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UKAbstractIncreased risk and severity of idiopathic pulmonary arterial hypertension (iPAH) is associated with elevated estradiol in men andpostmenopausal women. Pulmonary arteries synthesise estradiol via aromatase and metabolise it via CYP1B1 to mitogenicmetabolites; SNPs in aromatase and CYP1B1 have been associated with PAH. This suggests that estradiol metabolism could bealtered in iPAH. This proof-of-concept study profiles estradiol and several metabolites of estradiol simultaneously in serum fromiPAH patients and controls. We show that the estradiol and metabolite profile is altered in iPAH and that 16-hydroxyestrone and16-hydroxyestradiol accumulate in iPAH patients with 16-hydroxyestrone levels relating to disease severity.Keywordspulmonary hypertension experimental, metabolism, sex, pulmonary hypertensionDate received: 28 October 2019; accepted: 1 February 2020Pulmonary Circulation 2020; 10(1) 1–5DOI: 10.1177/2045894020908783IntroductionIncreased risk and severity of idiopathic pulmonary arterialhypertension (iPAH) is associated with elevated plasmaestradiol in both men and postmenopausal women,1,2 sug-gesting that estradiol is not ovarian-derived. Aromatase isresponsible for the synthesis of estradiol. Remodeled pul-monary arteries, lesions, and human pulmonary arterysmooth muscle cells (hPASMCs) from pulmonary arterialhypertension patients have high levels of aromatase.3Cytochrome P450 Family 1 Subfamily B Member 1(CYP1B1) can convert estradiol to mitogenic 16-hydroxyes-trogens and this enzyme is over-expressed in diseased pul-monary arteries from pulmonary arterial hypertension(PAH) patients.4 Existing assays have not been sensitive orspeciﬁc enough to detect levels of many estradiol metabol-ites in PAH patient blood. Using a newly developed liquidchromatography tandem mass spectrometry (LC-MS/MS)assay,5 here we investigate the proﬁle of several estradiolmetabolites in iPAH patients.MethodsSerum from 22 subjects with iPAH (12 males, 10 females)and 34 healthy control subjects (17 males, 17 females)were analyzed by LC-MS/MS for estrone (E1), 17a-estradiol (17aE2), 17-estradiol (E2), 16-hydroxyestrone(16OHE1), 16-hydroxyestradiol (16OHE2), 2-methoxyes-trone (2MeOE1), 4-methoxyestrone (4MeOE1), 2-methox-yestradiol (2MeOE2), and 4-methoxyestradiol (4MeOE2).5Data were compared by a Mann–Whitney U test with asso-ciations assessed using Pearson’s correlations. Values belowthe conﬁrmed limit of quantiﬁcation (LOQ) were imputed asthe LOQ (2 pg/mL E1, E2, 16OHE2, 2MeOE1, and 6 pg/mL16OHE1).Corresponding author:Margaret R. MacLean, Strathclyde Institute of Pharmacy and BiomedicalSciences, University of Strathclyde, HW406, The Hamnett Wing, 161,Cathedral Street, Glasgow G4 0RE, UKEmail: mandy.maclean@strath.ac.ukCreative Commons CC-BY: This article is distributed under the terms of the Creative Commons Attribution 4.0License (http://creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of thework without further permission provided the original work is attributed as specified on the SAGE and Open Access pages(https://us.sagepub.com/en-us/nam/open-access-at-sage).! The Author(s) 2020.journals.sagepub.com/home/pulTo determine if 16OHE2 was present at bioactiveconcentrations, proliferation was studied in hPASMCs andcell migration was studied in human blood outgrowthendothelial cells (BOECs). hPASMCs were isolated fromdistal pulmonary arteries (0.3–1mm diameter) frommacroscopically normal controls or patients undergoinglung transplantation. BOECs were isolated from PAHpatients as described previously6 with cells cultured asdescribedpreviously.7Thepatientsweremale and female (her-itable PAH/iPAH). Cells were stimulated with 16OHE2 fol-lowing preliminary concentration-response analysis (10 nMhPASMCs and 1 nM BOECs) for 48 h in phenol red-freemedia. Cell counts and cell migration (Corning Transwellinserts) were assessed using a Countess II FL cell counter(Life Technologies, UK). A student’s t-test was applied forcomparison of 16OHE2 vs vehicle-treated cells for eachgroup.ResultsE1 was decreased in serum of female iPAH patients whilst16OHE2 was increased. E1, E2, and 16OHE1 were elevatedin male iPAH patients (Table 1). Comparing males andfemales, E1 was higher in female controls compared withmale controls (p¼ 0.01). In iPAH, E1 was higher in malesthan females (p¼ 0.03).In female iPAH patients, increased E1 was associatedwith increasing pulmonary artery wedge pressure (PAWP)(R2¼ 0.5, ¼ 0.7, p¼ 0.03), decreased E2 was associatedwith increasing age (R2¼ 0.7, ¼ –0.8, p¼ 0.02), andincreasing E2 with increasing PAWP (R2¼ 0.6, ¼ 0.6,p¼ 0.05) while increased 16OHE1 correlated with a reduc-tion of cardiac output (R2¼ 0.7, ¼ –0.6, p¼ 0.01). In maleiPAH, increased E2 was associated with increased PAWP(R2¼ 0.5, ¼ 0.6, p¼ 0.02) while increased E1 and E2 asso-ciated with increasing BNP levels (R2¼ 0.6, ¼ 0.8, p¼ 0.01and R2¼ 0.4, ¼ 0.6, p¼ 0.04).16OHE2 caused proliferation of hPASMCs from femalePAH patients (by 63%) but not controls (male or female)(Fig. 1a–c). 16OHE2 caused increased migration of BOECsfrom PAH patients, both in males (by 55%) and females(by 67%) (Fig. 1d).DiscussionThis study is the ﬁrst to simultaneously quantify severalestrogens in controls and PAH patients as our methodo-logical paper measured levels in pooled plasma samples.5LC-MS/MS allowed discrimination between isomers of E2and its bioactive metabolites such as 16OHE1, 16OHE2,and methoxyestrogens. Increased 16-hydroxylation of E1/E2 promotes formation of proliferative metabolites and weidentiﬁed accumulation of 16OHE2 in female iPAH patientserum for the ﬁrst time. We conﬁrmed increased levels of16OHE1 in patients which had been suggested by previousstudies.8,9 In female iPAH, 16OHE2 was also increased.There was increased E1 and E2 levels in male iPAH patients,consistent with a previous study using immunoassay detec-tion.1 The E2 concentrations we determined here were lowercompared with previous studies determined using immuno-assays. This is consistent with immunoassays being limitedby cross-reactivity and over-estimation of E2 levels com-pared with LC-MS/MS.10Aromatase andCYP1B1 are increased in PAHpatient pul-monary arteries and pulmonary vascular lesions.3,4 Increasedaromatase activity may account for increased plasma E2 inmen and post-menopausal women with iPAH.1,2 In femaleiPAH, there was a decrease in E1 with no change in E2. Thechange in E1 may reﬂect an increased conversion to 16OHEsvia CYP enzymes as reﬂected by increased 16OHE1 and16OHE2 in these patients. Where there is a decrease in E1with no change in E2, this could also be due to the increasedmetabolism of E2. Baird et al. recently demonstrated that E2levels are elevated in post-menopausal iPAH female patientscompared with controls; however, E2 levels from controls inthat study were 5–7 pg/mL, commensurate with post-meno-pause levels.2 The majority of females in this current studywere pre-menopausal, with sampling not routinely timed toconsidermenstrual cycle, andmeanE2 concentrations in con-trols and patients were 34 pg/mL. Hence, it is perhaps unli-kely that a further increase would be expected in this patientcohort. Future clinical studies could consider blood samplingfrom pre-menopausal women at the same stage of the men-strual cycle.3As 16OHE1 can be derived directly from E1 andmale patients have higher E1, this may also contribute tothe elevated 16OHE1 in these patients. Consistent withincreased 16OHEs observed in iPAH patients, polymorph-isms in CYP1B1 are observed in PAH patients associatedwith functional diﬀerences in 16-hydroxylation, cancerrisk, and E2-mediated carcinogenicity.11,12 Conversionof E2 to 16OHE1 by CYP1B1 may contribute to thepathogenesis of experimental pulmonary hypertensionand cause proliferation of hPASMCs via increased oxidativestress.7,8We have already demonstrated that 16OHE1 can causeproliferation of both male and female hPASMCs and malemouse PASMCs at concentrations observed in plasma.4,7,1316OHE1 acts through the ERa receptor in hPASMCs7,14and indeed we have shown that ERa receptors are morehighly expressed in female PAH hPASMCs.15 This is con-sistent with female hPASMCs being more proliferative toE2, also due to decreased basal bone morphogenetic proteinreceptor II (BMPRII) signaling in female hPASMCs.1616OHE2 is a novel metabolite with unknown eﬀects thatwas increased in the serum from female iPAH patients com-pared with controls. This could also be due to alteredCYP1B1-mediated 16-hydroxlation.4,13 16OHE2 is an E2-receptor agonist, capable of cellular proliferation associatedwith cancer risk and E2-mediated carcinogenicity.12Consistent with this, we now show that, at a pathophysio-logical concentration, 16OHE2 can also induce proliferation2 | Estrogen metabolites in a small cohort of patients with iPAH Denver et al.in female human PAH PASMCs with no eﬀect in female ormale control PASMCs where ERa expression is not so high.However, we show that 16OHE2 increases cell migration inboth male and female PAH BOECs. As 16OHE1 has beenshown to act as a ‘‘second hit’’ to uncover a pulmonaryhypertensive phenotype in BMPRII mutant male andfemale mice,9 this suggests that both 16OHE1 and16OHE2 may also facilitate pulmonary vascular remodelingTable 1. Clinical characteristics and estrogen concentrations for controls vs iPAH patients.Female controls Female iPAH P-Values Male controls Male iPAH P-ValuesSubjects 17 10 17 12Clinical characteristicsAge (y) 39.0 (31–45) 56.5 (43–61) 0.01 32.5 (27–37) 57.0 (50–62) 0.001BMI (kgm–2) 24.9 (22–28) 29.1 (28–31) 0.06 23.8 (22–33) 32.8 (29–34) 0.07Right atrial pressure (mmHg) 6.0 (4–10) 9.0 (7–14) 0.23mPAP (mmHg) 55.0 (35–62) 51.5 (40–60) 0.97Cardiac output (L/min) 5.3 (5–7) 4.6 (4–6) 0.38PAWP (mmHg) 9.0 (8–12) 11.0 (9–16) 0.23PVR (mmHg/lmin) 9.0 (4–13) 9.1 (4–11) 0.97BNP (pg/mL) 76.0 (25–238) 114.0 (21–350) 0.79Current PAH medicationsERA 3 4PDE5 inhibitor 2 4Epoprostenol or treprostinil 2 2Calcium channel blocker – 1Treatment naı¨ve – 1Estrogen concentrations (pg/mL)E1 34.9 (27–75) (100) 16.9 (9–34) (91) 0.02 23.6 (17–32) (100) 28.1 (24–55) (100) 0.05E2 16.8 (10–59) (100) 7.9 (4–65) (100) 0.15 16.0 (10–21) (100) 21.3 (19–30) (100) 0.0216OHE1 24.8 (11–38) (65) 29.8 (17–53) (91) 0.09 24.1 (19–32) (47) 79.9 (58–96) (75) 0.0116OHE2 10.7 (9–11) (18) 14.6 (8–26) (82) 0.01 15.4 (8–18) (41) 15.9 (6–33) (67) 0.242MeOE1 8.4 (6–26) (41) 6.9 (6–80) (55) 1.00 10.9 (9–26) (30) 13.2 (8) –4MeOE1 ND ND – ND ND –2MeOE2 ND ND – ND ND –4MeOE2 ND ND – ND ND –<50 y estrogenconcentrations (pg/mL)n¼ 14 n¼ 4E1 33.7 (27–48) 39.5 (24–52) 1.00E2 24.0 (12–64) 47.7 (25–88) 0.5716OHE1 21.9 (12–35) 34.4 (16–52) 0.2316OHE2 10.1 (9–11) 14.0 (10–15) 0.062MeOE1 8.4 (7–18) 7.0 0.6450 y estrogenconcentrations (pg/mL)n¼ 3 n¼ 6E1 78.6 (56–107) 12.1 (9–19) 0.02E2 9.6 (7–13) 4.6 (3–7) 0.2616OHE1 37.7 29.8 (27–38) 0.3816OHE2 16.5 21.1 (9–28) 0.262MeOE1 16.0 (11–21) 6.5 (6–36) 0.90BMI: body mass index; ERA: endothelin receptor antagonist; PDE5: phosphodiesterase type 5 inhibitor; PAH: pulmonary arterial hypertension; iPAH: idiopathicPAH; mPAP: mean pulmonary artery pressure; PAWP: pulmonary artery wedge pressure; PVR: pulmonary vascular resistance; BNP: B-type natriureticpeptide; ND: not detected; E1: estrone; E2: 17-estradiol; 16OHE1: 16-hydroxyestrone; 16OHE2: 16-hydroxyestradiol; 2MeOE1: 2-methoxyestrone; 4MeOE1:4-methoxyestrone; 2MeOE2: 2-methoxyestradiol; 4MeOE2: 4-methoxyestradiol.Notes: Medication details of three female patients is unknown. Clinical characteristics and estrogen levels shown as median (Q1–Q3) (% LC-MS/MS detection).P-values following a Mann–Whitney U test in control vs iPAH or female iPAH vs male iPAH clinical characteristics.Pulmonary Circulation Volume 10 Number 1 | 3in males and females by acting as a ‘‘second hit’’ to BMPRIImutations. 16OHE2 is also produced in abundance duringpregnancy and it is interesting to speculate that it may con-tribute to pregnancy being a risk factor for PAH.17It should be noted that all of the medications that thepatients were prescribed are metabolized in the liver by CYPenzymes such as CYP3A4 and CYP2C9/8 and have noknown eﬀects on lung or liver CYP1B1 or CYP1A1 activity.One limitation of this study is that the patients were olderthan the controls. However E2 and 16OHE1 levels corre-lated with indicators of disease severity within the patientgroups and decreased E2 was associated with increasing agein iPAH patients. This suggests that increased E2/metabolitelevels in PAH patients are related to disease status ratherthan diﬀerence in age.The results suggest that estrogen metabolism isaltered in PAH and both 16OHE1 and 16OHE2 accumu-late, with 16OHE1 levels relating to disease severity.This study is limited by the small size but important as itwarrants further multi-center fully powered studies toconﬁrm and correlate metabolite levels with severity andsub-groups of PAH. Future studies on CYP enzymes andCYP1B1 inhibitors in patients with PAH may also bevaluable.Ethical approvalYes.GuarantorMargaret R. MacLean.Author contributionsNina Denver: all LC-MS/MS, data analysis, hPASMC experi-ments, and preparation of manuscript; Natalie Homer and RuthAndrew: analysis of LC-MS/MS; Katie Y. Harvey: BOEC experi-ments; Nicholas Morrell: isolation of BOECs; Eric D. Austin:blood sampling from all patients and controls; and Margaret R.MacLean: PI, grant holder, preparation of manuscript, and dataanalysis.Conflict of interestThe author(s) declare that there is no conﬂict of interest.FundingThe project described was supported by the British HeartFoundation, through grants RE/13/5/30177 and RG/16/2/32153,the Wellcome Trust (108468/z/15/z), the National Institutes ofHealth (R01HL134802 and P01HL108800), and a BBSRCiCASE PhD award (BB/N503691/1).Fig. 1. 16OHE2 stimulation increases proliferation and migration of human cells. 10 nM 16OHE2 increases proliferation in female PAH (a) withno effect on female control (b) and male control (c) human PASMCs. 1 nM 16OHE2 increased migration of male and female BOECs derived fromPAH patients (d).Veh: Vehicle control, Ethanol; 16OHE2: 16-hydroxyestradiol; PAH-BOECs: Blood outgrowth endothelial cell from pulmonary arterial hyperten-sion patients.Note: Data shown as mean SEM (n¼ 4–6) with statistics performed by comparison of 16OHE2 to veh by a Student’s t test *p< 0.05.4 | Estrogen metabolites in a small cohort of patients with iPAH Denver et al.References1. Ventetuolo CE, Baird GL, Barr RG, et al. Higher estradiol andlower dehydroepiandrosterone-sulfate levels are associated withpulmonary arterial hypertension in men. Am J Respir Crit CareMed 2016; 193: 1168–1175.2. Baird GL, Archer-Chicko C, Barr RG, et al. Lower DHEA-Slevels predict disease and worse outcomes in post-menopausalwomen with idiopathic, connective tissue disease- and congeni-tal heart disease-associated pulmonary arterial hypertension.Eur Respir J 2018; 51: pii: 1800467.3. Mair KM, Wright AF, Duggan N, et al. Sex-dependent influ-ence of endogenous estrogen in pulmonary hypertension. Am JRespir Crit Care Med 2014; 190: 456–467.4. White K, Johansen AK, Nilsen M, et al. Activity of the estro-gen-metabolizing enzyme cytochrome P450 1B1 influences thedevelopment of pulmonary arterial hypertension/clinical per-spective. Circulation 2012; 126: 1087–1098.5. Denver N, Khan S, Stasinopoulos I, et al. 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Estrogen metabolites in a small cohort of patients with idiopathic pulmonary arterial hypertension.

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