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Role of Estradiol Metabolism in the Pathophysiology and Pharmacology of Estrogen Therapy

English title Role of Estradiol Metabolism in the Pathophysiology and Pharmacology of Estrogen Therapy
Applicant Dubey Raghvendra K.
Number 138067
Funding scheme Project funding (Div. I-III)
Research institution Klinik für Reproduktions-Endokrinologie Departement Frauenheilkunde Universitätsspital Zürich
Institution of higher education University of Zurich - ZH
Main discipline Molecular Biology
Start/End 01.01.2012 - 30.09.2015
Approved amount 560'000.00
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Lay Summary (English)

Lead
Lay summary

Onset of menopause is accompanied with ovarian dysfunction, reduction in estradiol production, and initiation of multiple disorders, including coronary artery disease the leading cause of mortality in women. Since estrogen replacement therapy has protective biological effects, it is hypothesized that estrogens play a very important role in preventing diseases associated with menopause, such as coronary artery disease. With the increase in life expectancy, the number of postmenopausal women in the modern world including Switzerland, will triple by the year 2020. Because, all women will eventually reach menopause, the ailments associated with menopause will have to be treated and hormone replacement treatment which will inevitably play a key therapeutic role in treating these patients. Although, estrogen prevents cardiovascular disease in postmenopausal women, only 50% of the PMW benefit from and the mechanisms by which it induces its effects are still undefined. The experiments proposed in the present study will help us define the exact mechanism by which estradiol protects against cardiovascular disorders. If 17b-estradiol metabolites (e.g. 2-methoxyestradiol) are cardioprotective, then non-estrogenic 17b-estradiol metabolites could be employed for prevention of cardiovascular disease in both men and women without increasing the risk of cancer. Also, our hypothesis would explain the variable response to estrogens in postmenopausal women and would predict that therapy with 17b-estradiol metabolites would afford cardiovascular protection regardless of inter-individual differences in 17b-estradiol metabolism. Moreover, the use of non-feminizing estradiol metabolites may be a key therapeutic approach to prevent cardiovascular disease in both men and women. Finally, the studies with the metabolites will help us develop new therapeutic strategies for HRT to prevent cardiovascular disease.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Natural and environmental oestrogens induce TGFB1 synthesis in oviduct cells
Cometti Barbara P S, Dubey Raghvendra K, Imthurn Bruno, Rosselli Marinella (2018), Natural and environmental oestrogens induce TGFB1 synthesis in oviduct cells, in Reproduction, 155(3), 233-244.
Dihydrotestosterone induces pro-angiogenic factors and assists homing of MSC into the cardiac tissue
Popa Mirel-Adrian, Mihai Maria-Cristina, Constantin Alina, Şuică Viorel, Ţucureanu Cătălin, Costache Raluca, Antohe Felicia, Dubey Raghvendra K, Simionescu Maya (2018), Dihydrotestosterone induces pro-angiogenic factors and assists homing of MSC into the cardiac tissue, in Journal of Molecular Endocrinology, 60(1), 1-15.
2-MethoxyestradiolA 17β-Estradiol Metabolite With Gender-Independent Therapeutic Potential
Dubey Raghvendra K. (2017), 2-MethoxyestradiolA 17β-Estradiol Metabolite With Gender-Independent Therapeutic Potential, in Hypertension, 69(6), 1014-1016.
The estrogen metabolites 2-methoxyestradiol and 2-hydroxyestradiol inhibit endometriotic cell proliferation in estrogen-receptor-independent manner
Samartzis Eleftherios P., Imesch Patrick, Twiehaus Anja, Dubey Raghvendra K., Leeners Brigitte (2016), The estrogen metabolites 2-methoxyestradiol and 2-hydroxyestradiol inhibit endometriotic cell proliferation in estrogen-receptor-independent manner, in Gynecological Endocrinology, 32(7), 529-533.
2-Methoxyestradiol, an endogenous 17β-estradiol metabolite, inhibits microglial proliferation and activation via an estrogen receptor-independent mechanism
Schaufelberger Sara A., Rosselli Marinella, Barchiesi Federica, Gillespie Delbert G., Jackson Edwin K., Dubey Raghvendra K. (2016), 2-Methoxyestradiol, an endogenous 17β-estradiol metabolite, inhibits microglial proliferation and activation via an estrogen receptor-independent mechanism, in American Journal of Physiology-Endocrinology and Metabolism, 310(5), E313-E322.
Role of G-protein Coupled Estrogen Receptor in Mediating the Vasoprotective Actions of Estradiol
UnterleutnerElisabeth (2016), Role of G-protein Coupled Estrogen Receptor in Mediating the Vasoprotective Actions of Estradiol, ETH Zurich Library, Zurich.
2-Methoxyestradiol blocks the RhoA/ROCK1 pathway in human aortic smooth muscle cells
Rigassi Lisa, Barchiesi Bozzolo Federica, Lucchinetti Eliana, Zaugg Michael, Fingerle Jürgen, Rosselli Marinella, Imthurn Bruno, Jackson Edwin K., Dubey Raghvendra K. (2015), 2-Methoxyestradiol blocks the RhoA/ROCK1 pathway in human aortic smooth muscle cells, in American Journal of Physiology-Endocrinology and Metabolism, 309(12), E995-E1007.
Defining the Role of Androgens in Vascular Remodeling Associated with Cardiovascular Disease
Yulia Plutino (2015), Defining the Role of Androgens in Vascular Remodeling Associated with Cardiovascular Disease, ETH E-collection ETH Institutional Repository, Swiss Federal Institute of Tschnology Zurich.
17Beta-Estradiol Stimulated Integration of Human Mesenchymal Stem Cells in Heart Tissue
Mihai Cristina, Popa Mirel-Adrien, Viorel Julian, Antohe Felicia, Jackson Edwin, Simionescu Maya, Dubey Raghvendra, 17Beta-Estradiol Stimulated Integration of Human Mesenchymal Stem Cells in Heart Tissue, in Journal of Molecular and Cellular Cardiology, 133, 115-124.
Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D
Dubey Raghvendra, Fingerle J, Gillespie DG, Mi Z, Rosselli M, Imthurn B, Jackson EK, , Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D, in Hypertension, 66.

Collaboration

Group / person Country
Types of collaboration
Prof. Edwin K Jackson/University of Pittsburgh United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Bernhard Odermatt Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr. Juergen Fingerle/Hoffmann La Roche Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
American Heart Association Council on Hypertension Poster G-protein coupled estrogen receptor stimulates capillary formation by human umbilical vein endothelial cells via Alk1-SMAD1/5/8 pathway activation 16.09.2015 Washington DC, United States of America Unterleutner Elisabeth; Barchiesi Bozzolo Federica; Dubey Raghvendra K.;
American Heart Association Council on Hypertension Poster Inhibition of MicroRNA-221 by Estradiol Contributes to its Differential Effects on Smooth Muscle Cell Growth and Endothelial Cell Capillary Formation 16.09.2015 Washington DC, United States of America Unterleutner Elisabeth; Barchiesi Bozzolo Federica; Dubey Raghvendra K.;
11th Symposium of the Zurich Center for Integrative Human Physiology Poster Differential Role of AKT-Pathway in mediating the growth Effects of GPER in HCSMCs and HUVECs 21.08.2015 Zurich, Switzerland Barchiesi Bozzolo Federica; Dubey Raghvendra K.; Unterleutner Elisabeth;
11th Symposium of the Zurich Center for Integrative Human Physiology Poster Role of microRNA-221 in mediating the protective action of Estradiol in Vascular Cells 21.08.2015 Zurich, Switzerland Unterleutner Elisabeth; Barchiesi Bozzolo Federica; Dubey Raghvendra K.;
14th Day of Clinical Research Talk given at a conference Differential Role of AKT-pathway in Mediating the Growth Effects of GPER in Human Coronary Arterial Smooth Muscle Cells and Human Umbilical Vein Endothelial Cells 09.04.2015 Zurich, Switzerland Dubey Raghvendra K.; Unterleutner Elisabeth; Barchiesi Bozzolo Federica;
American Heart Association Arteriosclerosis Thrombosis and Vascular Biology Poster Capillary Formation and Autologus Regulation of Androgen Receptor Expression in Endothelial Progenitor Cells (EPCs) by DHT: Potential Implications for EPC Mediated Cardiovascular Repair in Men 09.09.2014 Toronto, Canada Dubey Raghvendra K.; Plutino Yuliya; Barchiesi Bozzolo Federica;
10th Symposium of the ZIHP Talk given at a conference G-Protein coupled Estrogen Receptor Mediates Capillary Formation 29.08.2014 Zurich, Switzerland Dubey Raghvendra K.; Unterleutner Elisabeth; Barchiesi Bozzolo Federica;
13th Day of Clinical Reseach Poster Activation of Intracellular Calcium Triggers DHT-induced pSMAD1/5/8 Signaling in EPCs 12.06.2014 Zurich, Switzerland Barchiesi Bozzolo Federica; Dubey Raghvendra K.; Plutino Yuliya;
13th Day of Clinical Research Poster G-protein Coupled Estrogen Receptor ediates Capillary Formation and Induces Vasculaogenic Pathway ALK1/pSMAD1/5/8 12.06.2014 Zurich, Switzerland Barchiesi Bozzolo Federica; Dubey Raghvendra K.; Unterleutner Elisabeth;
13th Day of Clinical Research Poster Regulation of microRNAs in Endothelial Progenitor Cells (EPCs) by Sex Steroids 12.06.2014 Zurich, Switzerland Dubey Raghvendra K.; Barchiesi Bozzolo Federica; Plutino Yuliya;
American Heart Association Arteriosclerosis Thrombosis and Vascular Biology Poster GPR30 Stimulates Capillary Formation by Human Endothelial Progenitor Cells 18.04.2012 Chicago, United States of America Barchiesi Bozzolo Federica; Dubey Raghvendra K.;


Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
10th Womens Health Conference, How Environment, Life style and Toxicants can Influence Womens Health Talk 09.01.2014 Geneva, Switzerland Dubey Raghvendra K.;


Awards

Title Year
Best poster Award 2014

Associated projects

Number Title Start Funding scheme
64040 Estrogen(s) and cardiovascular protection in postmenopausal women: Defining the cellular and biochemical mechanisms. 01.10.2001 Project funding (Div. I-III)
106098 Role of Estradiol Metabolism in the Pathophysiology and Pharmacology of Estrogen Therapy 01.10.2004 Project funding (Div. I-III)
117998 Role of estradiol metabolism in the pathophysiology and pharmacology of estrogen therapy 01.10.2007 Project funding (Div. I-III)

Abstract

Summary Hormone therapy (HT) with estrogen alleviates multiple postmenopausal disorders. Epidemiological/observational studies provide strong evidence the estrogens protect women against cardiovascular disease, however, results from two randomized clinical trials with conjugated equine estrogens (HERS and WHI study) showed no primary or secondary cardiovascular protective effects. However, another trial using estradiol (EPAT) demonstrated cardiovascular protective effects, additionally, protective effects were also observed in a sub-population of PMW who initiated HT within 10 years of menopause. The differences in the outcome of these studies remain controversial. Because the mechanisms by which estrogens induce their cardiovascular protective and/or deleterious effects are unclear and several different types of estrogens are used clinically it is hard to explain the reasons for these differential effects and underscores the need to explore basic mechanisms of cardiovascular protection by estrogens. Ongoing studies from our laboratory demonstrate that 2-methoxyestradiol (2-ME), a major metabolite of 17ß-estradiol with no binding affinity for estrogen receptors (ERs), is more potent than 17ß-estradiol in inhibiting vascular smooth muscle cell (SMC) and cardiac fibroblast (CF) growth. These observations, together with the findings that estradiol inhibits injury-induced neointima formation in mice lacking the functional ER-a, ER-ß, or both ER-a and ER-ß, led us to hypothesize that in addition to ERs, estradiol metabolites, are also responsible for the cardiovascular protection afforded by estrogen therapy and that variable metabolism of estrogen(s) explains the inconsistent clinical response to HRT. In this context using molecular (siRNA; knock out mice) and pharmacological approaches we previously demonstrated that ER-independent anti-proliferative effects of estradiol is mediated via its sequential conversion to methoxyestradiols. Because, 2-ME is anticarcinogenic it may serve as a safer alternative for HT. Hence using cellular (vascular cells and bone marrow derived progenitor cells), molecular (gene silencing, microarrays) and biochemical approaches, the objective of this proposal is to investigate the mechanisms by which estradiol, 2-ME and estradiol-derived 2-ME influences key mechanisms associated with the pathophysiology of vascular remodeling process in coronary artery disease (CAD) and atherosclerosis. We have previously shown that sequential metabolism of estradiol to methoxyestradiol by CYP450 and COMT is responsible for its antimitogenic actions on vascular SMCs. However, in depth mechanisms via which 2-ME inhibits vascular remodeling process associated with CAD/atherosclerosis is still lacking. Hence in this proposal, using molecular, biochemical and pharmacological techniques in cultured human bone marrow derived progenitor-SMCs (contributes to neointima formation by 66%), and progenitor-endothelial cells (PECs), and SMCs we will evaluate the impact of estradiol, 2-ME and estradiol-derived 2-ME on key pathways associated with cardiovascular remodeling process in CAD/atherosclerosis. In this context the influence of estradiol, 2-ME or estradiol-derived 2-ME intracellular mechanisms regulating progenitor-SMC and SMC-proliferation, apoptosis and PECs-induced neo-vascularization will be assessed. As an extension of our previous findings that estradiol, 2-ME and estradiol-derived 2-ME inhibits SMC growth by interfering with pathways regulating cell cycle, using gene silencing/pharmacological inhibitors, we will study the role of sequential metabolism of estradiol to 2-ME in inhibiting signal transduction pathways in progenitor-SMCs by assaying the effects on the mitogen-activated protein kinase (ERK1/2), the protein kinase C (PKC) pathway, Akt-phosphorylation, cyclin dependent kinases (cdc2/cdk1, cdk2); cyclins A and E, and tubulin polymerization, and cdk-inhibitors (p21/p27). Moreover, we will study whether estradiol and 2-ME induce apoptosis in Progenitor-SMCs by utilizing TUNNEL assay, DNA fragmentation, and modulatory effects on various extrinsic and intrinsic apoptotic pathways (mitochondrial release of cytochrome c; disappearance of 21KDa Bid protein; cleavage of procaspase 3, 8, and 9; oxidative stress-H202 release; stress activated c-jun NH2 terminal [JNK-P]; Fas ligand and Fas associated death domain protein [FADD]). We recently showed that 2-ME lowers cholesterol levels, and downregulates genes for cholesterol synthesis pathway and Rho-pathway in SMCs. Since estradiol and 2-ME inhibit cholesterol-induced atherosclerosis, and activation of Rho-pathway induces CAD; we will investigate the mechanism(s) via which estradiol and 2-ME influence cholesterol synthesis and the Rho-kinase pathway in SMCs and progenitor-SMCs. Growth of ECs and PECs in response to estradiol helps in vascular recovery. We recently showed that estradiol stimulates capillary formation by PECs via a membrane ER linked to ER-?. Since GPR30 is a new membrane bound ER, we will study its role in mediatig estradiol-induced capillary formation. Additionally, the impact of 2-ME on the growth of PECs which contribute to capillary formation in plaques will be assessed. Finally, using human micro-array gene chips we will screen and document the early and late genes that are influenced by estradiol and 2-ME and subsequently analyze their role in mediating the antivasoocclusive actions.Potential Implications: If our hypothesis is correct and the estrogen metabolites are cardioprotective, then non-estrogenic and non-carcinogenic estrogen metabolites, such as 2-ME, could be employed for prevention of cardiovascular disease in both men and women without increasing the risk of cancer. Also, our hypothesis would explain the variable response to estrogens in PMW and would predict that therapy with estrogen metabolites would afford cardioprotection regardless of inter-individual differences in estrogen metabolism.
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