Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner
- Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, France
- CARFI facility, Angers University, France
- INSERM U1297, Paul Sabatier University (Toulouse III) , University Hospital (UHC) of Toulouse, France
- University Hospital (CHU) of Angers, France
- Groupe Interdisciplinaire de Génoprotéomique Appliquée, Université de Liège, Belgium
- INSERM U1085, IRSET (Institut de Recherche en Santé, Environnement et Travail), University of Rennes, France
Figures
Figure 1
Involvement of ERα in flow-mediated dilation (FMD).
FMD was measured in mesenteric resistance arteries isolated from male mice lacking ERα (Esr1-/-) and male wild-type littermates (Esr1+/+) (A). (B) FMD was determined in response to stepwise …
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Figure 1—source data 1
Data and statistical analysis from experiments plotted in Figure 1B—M.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig1-data1-v2.xlsx
Figure 2
Involvement of ERβ and endothelial ERα in flow-mediated dilation (FMD).
(A to D) FMD, precontraction, basal diameter and acetylcholine-mediated dilation measured in male mice lacking ERβ (Esr2-/-) and their littermate control (Esr2+/+). (E to H) FMD, precontraction, …
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Figure 2—source data 1
Data and statistical analysis from experiments plotted in Figure 2A—H.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig2-data1-v2.xlsx
Figure 3 with 1 supplement
Flow-mediated dilation in mice lacking nuclear or membrane-associated ERα.
Esr1 expression level in aortic endothelial cells (expression relative to the housekeeping genes Gapdh, Hprt and Gusb), flow-mediated dilation (FMD) acetylcholine-mediated dilation were measured in …
Figure 3—figure supplement 1
Markers of endothelial and smooth muscle cells in mouse aortic endothelial cells.
Data and analysis in Figure 3—figure supplement 1—source data 1.
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Figure 3—figure supplement 1—source data 1
Data and statistical analysis from experiments plotted in Figure 3B–D,F–H, and J–L and for Figure 3—figure supplement 1A–F.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
with one supplement: Effect of the blockade of NO synthesis and cyloxygenase on flow-mediated dilation.
Flow-mediated dilation (FMD) was determined in pressurized mesenteric resistance arteries isolated from male Esr1+/+ and Esr1-/- (A), C451A-WT and C451A-ERα (B), R264A-WT and R264A-ERα (C), AF20WT …
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Figure 4—source data 1
Data and statistical analysis from experiments plotted in Figure 4A–H.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Effect of the blockade of NO synthesis (L-NNA), cyclooxygenase (indomethacin) and EETs production (MSPPOH) on flow-mediated dilation.
Data and analysis in Figure 4—figure supplement 1—source data 1.
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Figure 4—figure supplement 1—source data 1
Data and statistical analysis from experiments plotted in Figure 4—figure supplement 1A–G.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig4-figsupp1-data1-v2.xlsx
Figure 5
eNOS and Akt phosphorylation in response to flow in perfused isolated mesenteric resistance arteries.
As illustrated on the scheme shown on the top of the figure, mesenteric resistance arteries were cannulated in vitro on glass micropipettes and perfused with physiological salt solution. Flow (50 …
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Figure 5—source data 1
Data and statistical analysis from experiments plotted in Figure 5A–I.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig5-data1-v2.xlsx
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Figure 5—source data 2
All the blots for Figure 5.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig5-data2-v2.pdf
Figure 6
Isolated and perfused kidney from C451A-ERα mice.
In the isolated and perfused kidney (A), the flow-pressure relationship was determined in C451A-ERα and WT mice (B). (C) Acetylcholine (1 µM)-mediated relaxation. The levels of nitrate-nitrite (D), …
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Figure 6—source data 1
Data and statistical analysis from experiments plotted in Figure 6B–F.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig6-data1-v2.xlsx
Figure 7 with 3 supplements
Flow-mediated dilation and oxidative stress.
Flow-mediated dilation was determined in mesenteric resistance arteries isolated from male WT and C451A-ERα mice before and after addition of PEG-SOD and catalase (SOD-catalase, A and B), catalase (C…
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Figure 7—source data 1
Data and statistical analysis from experiments plotted in Figure 7A–L.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Gene expression profile in the mesenteric isolated from mice lacking membrane-ERα.
Data and analysis in Figure 7—figure supplement 1—source data 1.
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Figure 7—figure supplement 1—source data 1
Data from experiments plotted in Figure 7—figure supplement 1A–X and in Figure 7—figure supplement 2A–T.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig7-figsupp1-data1-v2.xlsx
Figure 7—figure supplement 2
Gene expression profile in the mesenteric isolated from mice lacking membrane-ERα.
Figure 7—figure supplement 3
Mechanosensitive channels and ATP in FMD.
Data and analysis in Figure 7—figure supplement 3—source data 1.
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Figure 7—figure supplement 3—source data 1
Data and statistical analysis from experiments plotted in Figure 7—figure supplement 3A–C.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig7-figsupp3-data1-v2.xlsx
Figure 8
FMD after antioxidant treatments in mice lacking membrane-ERα.
FMD was determined in mesenteric resistance arteries isolated from male WT and C451A-ERα mice treated for 2 weeks with the anti-oxidant TEMPOL (A to F) or with a combination of vitamin E and vitamin …
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Figure 8—source data 1
Data and statistical analysis from experiments plotted in Figure 8A–L.
- https://cdn.elifesciences.org/articles/68695/elife-68695-fig8-data1-v2.xlsx
Figure 9
Schematic representation of the known E2-mediated ERα-dependent protective effects (upper panel) and of the new pathways described in the present study (lower panel).
Previous works (upper panel) have demonstrated the role of E2 and the nuclear activating function AF2 of ERα against atherosclerosis and hypertension (Guivarc’h et al., 2018) as well as in …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background(Mus musculus, males and females) | Esr1-/-C57BL/6 J(Symbol: Esr1tm1.1Mma, Synonyme: ERalpha Knockout) | Mouse Clinical Inst., Strasbourg, France, Dupont et al., 2000 | MGI:2386760 | |
| Strain, strain background(Mus musculus, males) | AF2°ERα,C57BL/6 J(Symbol: Esr1tm1.1Ohl Synonym: ERalpha-AF2°) | Mouse Clinical Inst., Strasbourg, France, Billon-Galés et al., 2009 | MGI:4950046 | |
| Strain, strain background(Mus musculus, males) | C451A-ERα, C57BL/6 N(Symbol: Esr1tm1.1Ics Synonyme: C451A-ERalpha knock-in) | Mouse Clinical Inst., Strasbourg, France, Adlanmerini et al., 2014 | MGI:5574591 | |
| Strain, strain background(Mus musculus, males) | TekCre/+:Esr1f/f, C57BL/6(B6.Cg-Tg(Tek-cre)12Flv/J backcrossed with Esr1tm1.2MmaSynonym:Tie2Cre ERαlox/lox) | Esr1lox/lox: Mouse Clinical Institut, Strasbourg, France.TekCre: Jackson Lab (Bar Harbor, Me), Billon-Galés et al., 2009TekCre:Koni et al., 2001Esr1lox/lox:Dupont et al., 2000 | TekCre:Esr1lox/lox: | MGI:3775510 |
| Strain, strain background(Mus musculus, males) | Esr2-/-,C57BL/6 J (Symbol: Esr2tm1MmaSynonym: ERbeta) | Mouse Clinical Inst., Strasbourg, France, Dupont et al., 2000 | MGI:2386761 | |
| Strain, strain background(Mus musculus, males) | R264A-ERα, C57BL/6 N | Mouse Clinical Inst., Strasbourg, France, Adlanmerini et al., 2020 | No MGI ID yet | |
| Antibody | Anti-eNOS, (mouse monoclonal, clone3) | BD Biosciences | Cat# 610297, RRID:AB_397691 | WB (1:1000) |
| Antibody | Anti-phospho-eNOS, pS1177 (Mouse monoclonal,Clone 19/eNOS/S1177) | BD Biosciences | Cat# 612392, RRID:AB_399750 | WB (1:1000) |
| Antibody | Anti-beta-actin, (Mouse monoclonal, clone AC-74) | Sigma-Aldrich | Cat#: 5316; RRID:AB_476743 | WB (1:5000) |
| Antibody | Anti-Akt Pan, (rabbit monoclonal, clone C67E7) | Cell signalling technologyOzyme | Cat#: 4691; RRID:AB_915783 | WB (1:1000) |
| Antibody | Anti-phospho-Akt, S473, (rabbit monoclonal, clone D9E) | Cell signalling technology Ozyme | Cat#: 4060; RRID:AB_2315049 | WB (1:2000) |
| Antibody | Anti-mouse IgG (H + L) Secondary antibody HRP (Goat polyclonal) | Thermo scientific | Cat#: 31430; RRID:AB_228307 | WB (1:5000) |
| Antibody | Anti-rabbit IgG(H + L) Secondary antibody HRP (Goat polyclonal) | Thermo scientific | Cat#: 31460; RRID:AB_228341 | WB (1:10000) |
| Chemical compound, drug | vitamin C | Sigma Aldrich Merck, Favre et al., 2011 | A5960 | |
| Chemical compound, drug | vitamin E | Sigma Aldrich Merck, Favre et al., 2011 | T3251 | |
| Chemical compound, drug | Mito-tempo | Sigma Aldrich Merck, Freed et al., 2014 | SML0737 | |
| Chemical compound, drug | catalase | Sigma Aldrich Merck, Bouvet et al., 2007 | C3155 | |
| Chemical compound, drug | PEG-superoxide dismutase (SOD) | Sigma Aldrich Merck, Bouvet et al., 2007 | S9549 | |
| Chemical compound, drug | Estetrol (E4) | Sigma Aldrich Merck, Abot et al., 2014 | SML1523 | |
| Chemical compound, drug | ICI 182 780 | Tocris Biotechne, Meyer et al., 2010 | 1047 | |
| Chemical compound, drug | G-1 ((±)–1-[(3aR*,4S*,9bS*)–4-(6-Bromo-1,3-benzodioxol-5-yl)–3 a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)]- ethanone | Cayman chemical Bertin Bioreagent, Meyer et al., 2010 | 10008933 | |
| chemical compound, drug | G-36 ((±)-(3aR*,4S*,9bS*)–4-(6-Bromo-1,3-benzodioxol-5-yl)–3 a,4,5,9b-tetrahydro-8-(1-methylethyl))–3H-cyclopenta[c]quinoline | Cayman chemical Bertin Bioreagent, Meyer et al., 2016 | 14,397 | |
| Sequence-based reagent | N-(methylsulfonyl)–2-(2-propynyloxy)-benzenehexanamide (MSPPOH) | Cayman chemical Bertin Bioreagent, Dietrich et al., 2009 | 75,770 | |
| Chemical compound, drug | Grammostola spatulata mechanotoxin 4 (GsMTx4) | Alomone Labs, John et al., 2018 | STG-100 | |
| Chemical compound, drug | YODA1 | Bertin Bioreagent, Lhomme et al., 2019 | SML1558 | |
| Chemical compound, drug | ATPγS | Tocris Biotechne, Kukulski et al., 2009 | 4080 | |
| Chemical compound, drug | 4-hydroxy-2,2,6,6-tetramethylpiperidine (TEMPOL) | Sigma Aldrich Merck, Freidja et al., 2014 | 176,141 | |
| commercial assay or kit | Nitric oxide metabolite detection kit | Cayman Chemical | 780,051 | |
| commercial assay or kit | Hydrogen peroxide assay kit | Abcam | Ab102500 | |
| commercial assay or kit | ATP determination kit | Invitrogen Molecular Probes | A22066 |
Additional files
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Supplementary file 1
Primer sequences used for the RT-qPCR.
- https://cdn.elifesciences.org/articles/68695/elife-68695-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68695/elife-68695-transrepform1-v2.docx
