Resistance arteries are the small blood vessels located upstream of capillaries. Alteration of their structures or functions can raise capillary pressure, which exacerbates organ damage due to cardio- and cerebro-vascular risk factors and associated organ disorders. The basal tone of resistance arteries allows for tight control of local blood flow. This tone results from the interaction between pressure-induced smooth muscle contraction and flow-mediated dilation (FMD) due to the activation of endothelial cells by shear stress. FMD measured in the human forearm depends mainly on the acute production of NO by endothelial cells in response to an acute increase in shear stress (Joannides et al., 1995; Green et al., 2014; Zhou et al., 2014) and reduced FMD is a hallmark of endothelium dysfunction (Zhou et al., 2014; Rizzoni and Agabiti Rosei, 2006; Stoner and Sabatier, 2012).

Epidemiological investigations have shown that, prior to menopause, women are less affected by cardiovascular disorders than men (Simoncini, 2009; Arnal et al., 2017). Estrogens protect against atherosclerosis (Billon-Galés et al., 2009) and neointimal proliferation (Smirnova et al., 2015), and accelerate re-endothelialization of injured arteries (Brouchet et al., 2001). Numerous actions of 17-beta-estradiol (E2) are mediated by estrogen receptor alpha (ERα), which acts in the nucleus as a transcription factor. E2 is strongly involved in the outward remodelling of the uterine blood vessels during pregnancy (Mandala and Osol, 2012). Indeed, we have previously shown that E2 and ERα, and more precisely its nuclear activating function AF2, are both essential for the arterial outward remodeling induced by a chronic rise in blood flow in vivo (Tarhouni et al., 2013; Tarhouni et al., 2014a; Tarhouni et al., 2014b).

However, a subpopulation of ERα is also associated with the plasma membrane and activates non-nuclear signaling (Arnal et al., 2017; Banerjee et al., 2014; Lu et al., 2017). The acute effect initially described in 1967 was a rapid increase in AMPc production in the rat uterus in response to E2 (Szego and Davis, 1967). E2 binding to the plasma membrane was subsequently reported in endometrial cells and hepatocytes (Pietras and Szego, 1977), suggesting that a fraction of ERα could be located to the membrane and contributes to the rapid effects of E2, possibly through the rapid activation of G proteins and kinases such as ERK1-2, PI3K, or P21ras (Arnal et al., 2017). In ovine fetal pulmonary artery endothelial cells, E2 stimulates eNOS activity through activation of ERα leading to increased intracellular Ca²⁺ within minutes (Lantin-Hermoso et al., 1997). By contrast, in HUVECs, E2 induces a rapid production of NO and cGMP independent of an increase in intracellular Ca²⁺ (Caulin-Glaser et al., 1997). This rapid nongenomic activation of eNOS involves Akt/PKB (Florian et al., 2004) and MAP kinase-dependent mechanisms (Chen et al., 1999). Estradiol-induced endothelium-independent dilation was also described in canine coronary arteries (Sudhir et al., 1995) and in rat cerebral microvessels (Florian et al., 2004). This dilation is also mediated by ERα located at the level of the plasma membrane. Using a mouse model lacking membrane-associated ERα, we demonstrated that the acute vasodilator effect of E2 and its accelerative effect on re-endothelialization are mediated by membrane-associated ERα (Adlanmerini et al., 2014; Zahreddine et al., 2021). On the other hand, E2 exerts protective effects against atheroma, angiotensin 2-induced hypertension, and neointimal hyperplasia through its nuclear effects (Guivarc’h et al., 2018).

The 7-transmembrane G-protein-coupled estrogen receptor (GPER, formerly known as GPR30) is another receptor located not only at the plasma membrane but also on the membrane of the endoplasmic reticulum that can be activated by E2. It was found in both human and animal arteries (Prossnitz and Barton, 2011; Barton et al., 2018). The combination of GPER-selective agonists and antagonists with the use of GPER-knock-out mice allowed to elucidate more specifically its biological effects arteries (Prossnitz and Barton, 2011; Barton et al., 2018). In the rat, GPER activation by its agonist G-1 reduces uterine vascular tone during pregnancy through activation of endothelium-dependent NO production (Tropea et al., 2015). Likewise, the G-1-induced relaxation of the mesenteric resistance arteries in both male and female rats is mainly mediated by the PI3K-Akt-eNOS pathway (Peixoto et al., 2017). Noteworthy, GPER partly contributes to E2-dependent vasodilation of mouse aortae (Fredette et al., 2018). Thus, both ERα and GPER could contribute to the rapid actions of E2, although their respective roles according to vessel type, species and pathophysiological context remain to be established.

The risk of cardiovascular diseases differs between men and women, and the protection of women is progressively lost after menopause. For instance, endothelium-dependent dilation of subcutaneous arteries is reduced in post-menopausal women compared to pre-menopausal women (Kublickiene et al., 2005; Kublickiene et al., 2008). This protection involves NO production in response to estrogens. Similarly, diet phytoestrogens could have protective actions in postmenopausal women suffering coronary artery disease (Cruz et al., 2008) and selective estrogen receptor modulators (SERMs) such as raloxifene exert protective actions in female rats through eNOS activation (Chan et al., 2010). Besides the activation of eNOS, hormonal replacement therapy also activates endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation as shown in rat mesenteric and uterine arteries (Burger et al., 2009) as well as in the rat gracilis muscle artery with increased Epoxyeicosatrienoic acids (EETs) production involved in E2-mediated increase in FMD in hypertensive or old rats (Huang et al., 2001; Sun et al., 2004). Furthermore, estrogen therapy reduces pressure (myogenic) (Kublickiene et al., 2005; Kublickiene et al., 2008) and adrenergic-dependent contraction (Meyer et al., 1997). FMD is also improved by E2 in rat gracilis muscle arteries (Huang et al., 1998). Although there is an increase in the amplitude of FMD in women among the menstrual cycle with a greater dilation during the luteal or follicular phase (Hashimoto et al., 1995), FMD is similar in healthy young men and women (Sullivan et al., 2015). Importantly, the first disruptive mutation in the gene encoding ERα, reported in 1994 in a man who was only 30 years old (Smith et al., 1994), was found to be associated with a total absence of FMD (Sudhir et al., 1997). This single yet major clinical observation suggests that ERα-dependent signal transduction could play a role in FMD in males. Of note, conversion of testosterone into estradiol by aromatase which is expressed in the arterial wall has been shown to reduce early atherogenesis in male mice (Nathan et al., 2001).

In the present study, we investigated the role of ERα and its different subfunctions on FMD in isolated mouse resistance arteries. To this aim, we used different mouse models that were: (i) fully deficient in ERα (Esr1^-/- mice), (ii) deleted in seven amino acid in the helix 12 and thus deficient in activation function (AF)–2 necessary for the nuclear transcriptional activity of ERα (AF2⁰ERα mice), and (iii) invalidated for plasma membrane-associated signaling. To explore the role of membrane ERα, we used: (1) mice that carry a mutation of the codon encoding the cysteine (Cys) 451 palmitoylation site of ERα to alanine (C451A-ERα mice) so that the anchoring of the receptor to the plasma membrane is impossible (Adlanmerini et al., 2014) and (2) a knock-in mouse model of ERα mutated for the arginine 264 (R264A-ERα mice) so that the interaction of the membrane-located ERα with Gαi involved in rapid NO production is suppressed (Adlanmerini et al., 2020). Moreover, because the absence of membrane-associated ERα strongly reduced FMD, additional experiments were conducted to investigate: (i) the involved mechanisms, (ii) its counterpart in female mice, and (iii) the potential role of ERα activation by its ligands.

We report here that endothelial membrane-associated ERα contributed to optimize flow (shear stress)-mediated dilation in young healthy mouse resistance arteries in a ligand-independent manner.

Previous experimental studies have reported the vascular benefit of estrogens on blood flow homeostasis, and E2 improves endothelium-dependent relaxation when it is reduced in diseased conditions. (Huang et al., 2001; Al-Khalili et al., 1998; Huang et al., 2000; Svedas et al., 2002; LeBlanc et al., 2009). Nevertheless, no difference in FMD has been observed between healthy men and women (Sullivan et al., 2015). In agreement, our results show that acute (20 min) incubation with exogenous E2, E4, or ICI-182780 did not affect FMD. Similarly, incubation with the GPER antagonist G-36 did not affect FMD, excluding ligand-activated GPER actions in FMD. In addition, endogenous estrogens in female WT mice had no impact on FMD as it was equivalent in male, female and ovariectomized female mice. These data suggest that FMD involves unliganded ERα activation in response to shear stress. In agreement, a recent study has reported another action of unliganded ERα, namely its inhibitory action on endothelial cell proliferation and migration (Lu et al., 2017).

Although FMD was reduced in Esr1^-/- mice, agonist-mediated endothelium dependent (acetylcholine and insulin) and independent (SNP) dilation was not affected suggesting a selective reduction in flow (shear stress)-dependent signaling without a change in receptor-dependent dilation in the endothelium and in the smooth muscle.

The present study also showed that FMD involves membrane-associated ERα. FMD was similarly reduced in Esr1^-/- mice and in both C451A-ERα and R264A-ERα mice. Although acute response (FMD and agonist-dependent dilation) can only be attributed to membrane-associated events, the expression level of the enzymes involved in the process could be modulated by the nuclear effects of ERα. Thus, endothelium-dependent dilation was investigated in mice lacking either the nuclear activating function AF2 of ERα or membrane-dependent action of ERα. Membrane-ERα is located at the level of the caveolae through either a binding to caveolin-1 or to striatin, thus creating a link with the Gαi and Gβγ proteins (Arnal et al., 2017). In order to abrogate the membrane effects of ERα, we used two different models. First, we used C451A-ERα mice that lack the palmitoylation site (cysteine at position 451) of the receptor so that the anchorage of ERα to the plasma membrane and the link to caveolin-1 is prevented (Adlanmerini et al., 2014). We also used a knock-in mouse model of ERα mutated for the arginine 264 (R264A-ERα mice) suppressing its interaction with Gαi involved in rapid eNOS activation (Adlanmerini et al., 2020). The fact that FMD was similarly reduced in Esr1^-/-, C451A-ERα, and R264A-ERα mice without change in receptor-dependent dilation, strongly supports that membrane-associated ERα is involved in FMD. This is further supported by the absence of reduction in FMD observed in AF2-ERα mice which only lack the AF2 nuclear function of ERα. AF2 is also involved in the vascular response to a chronic increase in flow (flow-mediated remodeling) which is absent in AF2⁰-ERα mice but fully present in C451A-ERα and R264A-ERα mice (Guivarc’h et al., 2018). This remodeling is a chronic adaptation of the vascular wall associated with changes in arterial diameter and wall mass within 2 weeks after a chronic rise in blood flow in vivo (Chehaitly et al., 2021). A chronic increase in blood supply, such as that needed for collateral growth in ischemic disorders, induces an increase in diameter together with wall thickening so that both shear and tensile stress are normalized within 1 week following the flow increase (Silvestre et al., 2001). This remodeling involves an early inflammatory phase allowing cell growth and reorganization in the arterial wall (Caillon et al., 2016) and a dilatory phase involving NO, prostaglandins and CO production (Dumont et al., 2007; Belin de Chantemèle et al., 2010; Freidja et al., 2011). Noteworthy, flow-mediated remodeling is absent in ovariectomized rats and mice and in Esr1^-/- mice (Tarhouni et al., 2013) whereas this remodeling is preserved in ovariectomized rats treated with E2 (Tarhouni et al., 2014b) or resveratrol (Petit et al., 2016). More recently, we have shown that this remodeling requires activation of AF2 and is independent on membrane-located ERα (Guivarc’h et al., 2018).

Another membrane receptor for E2 located at the plasma membrane is GPER (Prossnitz and Barton, 2011). Both ligand-dependent and ligand-independent activation of GPER have been reported (Meyer et al., 2016). GPER is involved in regulation of reproductive functions, endocrine regulation and metabolism, cardiovascular, kidney, neuroendocrine and cerebral functions function as well as immune cell function. Furthermore, previous studies suggest a role for GPER in hypertension, kidney diseases, diabetes, and immune diseases. Consequently, GPER is a potential therapeutic target for the treatment of these diseases (Prossnitz and Barton, 2011). In the present study, incubation with the GPER antagonist G-36 did not affect FMD, ruling out the role of ligand-activated GPER in FMD. However, a possible role of unliganded GPER activation cannot be excluded in case of a crosstalk between membrane-dependent ERα and GPER activation.

To characterize the effect of membrane-ERα on the NO pathway which is involved in FMD, we investigated the effect of L-NNA-mediated inhibition of NO-synthesis on FMD. L-NNA inhibited FMD in arteries from WT and AF2⁰ERα, but not arteries from Esr1^-/-, C451A-ERα and R264A-ERα mice, suggesting that membrane-associated ERα is involved in NO-dependent FMD. This is in agreement with a previous study that used the ERα Neo-KO model with incomplete deletion, thereby showing that the NO pathway (dilation sensitive to L-NAME) was reduced in response to flow in the gracilis artery of male mice (Sun et al., 2007). In contrast to FMD, L-NNA strongly reduced acetylcholine-mediated dilation in WT, Esr1^-/- AF2⁰ERα, C451A-ERα and R264A-ERα mice. Thus, the NO-pathway can be activated in response to receptor stimulation in C451A-ERα and R264A-ERα mice, whereas only its activation by flow was reduced in these mice lacking only membrane-ERα signaling. FMD can involve prostaglandins and EDHF such as EETs (Sun et al., 2007) and EDHF was shown to mediate estrogen-mediated dilation of the uterine arteries (Burger et al., 2009). Nevertheless, in the present study, cyclooxygenase inhibition with indomethacin and EETs production inhibition with MSPPOH did not affect FMD in both WT and Esr1^-/-, AF2⁰ERα, C451A-ERα and R264A-ERα mice, suggesting a limited role of the pathway in mesenteric arteries of male mice. Consequently, the remaining FMD following the addition of L-NNA, indomethacin and MSPPOH relies probably on other hyperpolarizing agents. Indeed, endothelium-dependent hyperpolarization (EDH) has a major role in resistance arteries homeostasis (Brandes et al., 2000; Garland and Dora, 2017) and COX-derivatives can also induce EDH in resistance arteries and in the carotid when submitted to flow (Ohlmann et al., 2005; Bergaya et al., 2001). In humans, FMD measured in the brachial artery relies mainly on the production of NO (Alexander et al., 2021). Furthermore, changes in FMD in the brachial artery predict well the endothelial dysfunction in human resistance arteries (Park et al., 2001). Nevertheless, the difference in the nature of the agents involved in FMD besides NO between humans and mice could be a limitation of the present study. As stated above, the involvement of EDH in FMD is greater in mouse resistance arteries than in humans when measured at the level of the brachial artery.

As FMD has a key role in blood flow delivery to organs (Hill et al., 2010), we investigated the flow-pressure relationship in the mouse kidney. In agreement with the reduction in FMD observed in resistance arteries, we found a leftward shift of the flow-pressure relationship in C451A-ERα mice further confirming the decreased sensitivity to flow of the resistance vasculature. Recent studies have shown that flow activates Piezo1-dependent release of ATP through pannexin hemi-channel followed by P2Y2 activation and NO production by endothelial cells (Wang et al., 2015; Wang et al., 2016). We found that both nitrate-nitrite and ATP productions were lower in the kidney perfusate from C451A-ERα than in WT mice, in agreement with the reduced NO-dependent FMD (sensitive to L-NNA) observed in isolated arteries. As in mesenteric arteries, we have previously shown that eNOS expression level in the kidney is not altered by the absence of membrane ERα in C451A-ERα (Guivarc’h et al., 2020). Although NO and ATP production were reduced in kidneys from C451A-ERα, the acute response to ATP and to the Piezo1 agonist YODA-1 was not affected by the absence of membrane ERα. In addition, the mechanosensitive channel blocker GsMTx4 similarly affected FMD in C451A-ERα and WT mice, suggesting that the defect in FMD associated with the absence of membrane ERα is probably located downstream flow sensing.

The reduction in NO-dependent FMD found in arteries from mice lacking membrane-ERα could be due to an excessive ROS production as the chronic treatment of C451A-ERα mice with an antioxidant treatment restored FMD to control level. In agreement, we found that H₂O₂ production by the kidney was higher in C451A-ERα than in WT mice. This observation is in agreement with a previous work that has shown that E2 increases the release of bioactive NO by inhibition of superoxide anion production in bovine endothelial cells (Arnal et al., 1996). Accordingly, we found that reducing total ROS or mitochondrial ROS production improved FMD in C451A-ERα mice. By contrast, catalase which eliminates H₂O₂ reduced FMD in C451A-ERα mice suggesting that an excessive ROS production due to the absence of membrane-associated ERα had a dual effect with (1) a reduction of NO bioavailability and (2) an increase in H₂O₂ production contributing to some vasodilating effect. Noteworthy, kidney perfusates showed higher levels of H₂O₂ in C451A-ERα mice. An excessive ROS production could also alter eNOS activation as previously shown through increased phosphatase activation (Ding et al., 2020). Previous studies have also shown that shear stress induces a more quiescent and less oxidative phenotype in endothelial cells (Doddaballapur et al., 2015; Wu et al., 2018), thus reducing the oxidative products of mitochondrial origin. Nevertheless, in a context of known dysfunctional FMD, a previous work has shown that H₂O₂ could mediate FMD in human coronary arteries from patients suffering coronary artery disease although H₂O₂ remains deleterious (Freed et al., 2014).

Conclusion

To conclude, these data demonstrate for the first time a major role of ERα, and more precisely of non liganded endothelial membrane-located ERα for optimal FMD and thereby a potential role in local blood flow homeostasis. The mechanism appears to involve an optimization of NO activation and/or a decrease in ROS production as depicted in Figure 9. The functional consequences in terms of arteriolar and tissue protection should now be investigated.

Figure 9

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All data generated or analysed during this study are included in the manuscript and supporting files. Source data files are provided for each figure and supplement.

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Decision letter after peer review:

Thank you for submitting your article "Membrane estrogen receptor alpha (ERα) optimizes flow-mediated dilation in both sexes, in a ligand-independent manner" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Philip W Shaul (Reviewer #4).

The reviewers have discussed their reviews with one another, and this letter is to help you prepare a revised submission.

Essential revisions:

1) The ligand-independent activation of membrane ERα is a novel finding. However, simply ignoring a potential role of GPER in such ligand-independent regulation of FMD is a major flaw. The authors should carry out some straight-forward studies to look at the effects of pharmacological inhibition using GPER antagonist, G-36, which turns out to not only inhibit GPER but also via inhibition of constitutive GPER expression, to reduce abundance (and activity) of Nox1. The authors are advised to perform in vitro studies of vessels from WT mice, evaluating dilation in response to flow +/- GPER antagonist, as long as a parallel control study is done evaluating dilation in response to GPER agonist +/- GPER antagonist.

2) In the Introduction section, the authors are encouraged to cite original works related to non-nuclear signaling by the subpopulation of ER associated with the plasma membrane in endothelium.

3) In the Introduction section, the background information about cardiovascular disease risk in men versus women and the impact of estrogens on risk is not as clear-cut as the authors imply. A more balanced presentation of the clinical evidence is warranted.

4) In the Introduction, a more detailed account of the published literature in the field including studies in humans should be provided The Authors may consider citing some studies from among PMID 19126786; 7642876; 20185791; 26471832; 9176294; 17367797; 28645860; 26734763; 9791075; 16131583; 18319309; 19086257; 9930647.

5) In the Results section, Page 6, line 106 incorrectly refers the reader to Figure 1B.

6) In the Results section. Page 6, line 117: fulvestrant is an ERα and ERβ antagonist (pKi = 9 for both) as well as a GPER full agonist (pKi = 7).

7) In the Results section, Figures 2B, 2F and 2J: ERα protein abundance in endothelial cells should be evaluated, even if only feasible from vasculature that would yield more endothelial cells than mesenteric resistance arteries.

8) In the Results section, Figure 5B: the flow-pressure relationship was shifted left, or up, in C451A-ERα mice versus WT according to the group labeling, and not to the right.

9) In the Results section Page 17, line 281: the use of the term autoregulation here may be confusing, and more specific interpretation would be helpful (and better placed in the Discussion section).

10) In the Results section, how does flow increase eNOS Ser1177 phosphorylation and alter H₂O₂ via ERα mechanistically ? Are flow-related changes in Akt phosphorylation in endothelium altered in the absence of ERα?

11) In the Results section, Do non-nuclear actions of plasma membrane-associated ERα influence endothelial cell production of prostaglandins in the setting of FMD?

12) In the Results section, do non-nuclear actions of plasma membrane-associated ERα influence mechanosensitive ion channel localization or function in endothelial cells?

13) In the Results section, why was the antioxidant TEMPOL specifically selected? Would the Authors expect a similar outcome following treatment with other antioxidants such as e.g. quercetin or dimethyl fumarate?

14) In the Results section, does Tempol or PEG-catalase acutely restore FMD ex vivo?

15) In the Results section, Line 312: Chronic for a 2-week treatment does not sound very appropriate.

16) In the Results section, flow rate may be changed to shear stress in Dyn/cm2: this would help comparing between published works in the topic.

17) The Discussion section should be shortened, providing more succinct focused discussion of the interpretation of the findings, their implications, and possible explanations to fill the new knowledge gaps that result from the work.

18) In the Conclusion section, Figure 7: the schematic is only in part helpful because how non-nuclear actions of membrane-associated ERα in endothelial cells govern eNOS and ROS in response to flow is not addressed.

Reviewer #1:

Flow mediated dilation (FMD) is a response to acute shear stress and its reduction is known to be a hallmark of endothelium dysfunction, associated with aging and with cardiovascular and metabolic disorder. Estrogen receptor alpha (ERα) was previously found to be associated with FMD in mouse and human. This manuscript further explore which ERα variant was important for FMD. The authors reported a novel ligand-independent pathway leading to flow mediated dilation attributed to the membrane-bound ERα. The authors used mouse model with fully deficient ERα, as well as mouse with specific inactivation of either membrane-bound or nuclear ERα and showed that only disruption of membrane-bound ERα greatly inhibit flow mediated dilation. The author further revealed the mechanism involving shear stress-induced attenuation of ROS levels which increase eNOS activity. Therefore, membrane-bound ERα could be a potential target therapy to reduce oxidative damage in endothelium of resistance arteries.

Strengths

This study presented for the first time that flow mediated dilation produced by estrogen receptor alpha is attributed to its membrane receptor via ligand-independent pathway.

Weaknesses

The mechanism of membrane receptor, ligand independent pathway of estrogen receptor alpha that the authors reported lacks novelty as it was already previously described.

Reviewer #2:

Multiple animal models have been used to test the contribution of membrane estrogen receptors on the vascular dilation induced by flow (flow-mediated dilation, FMD).

The authors propose that the presence of membrane estrogen receptors optimizes flow-mediated dilation. However, the conclusion that ER promotes NO production and inhibits oxidative stress is not fully supported by the data since basal ROS production is not altered in the models without functional ER, and an antioxidant treatment normalizes the dilatory response, and effect that can be independent of a direct NOS activation. The data show that in the absence of functional membrane receptors, flow-mediated dilation is reduced but that it is restored by a treatment with an antioxidant. Thus, these receptors seem not to be necessary for FMD. The involvement of these receptors in FMD remains therefore questionable.

Reviewer #3:

The Authors report that blood flow in mouse resistance arteries activates membrane-associated ERα, which reduces oxidative stress (O₂-.), resulting in enhanced NO bioavailability. The absence of membrane-associated ERα may lead to the production of O₂-. which attenuates NO-dependent dilation as shown by a rise in H₂O₂ generation in the perfused kidney.

Strengths

(1) Use of cutting-edge animal models to explore the role of ERα in the vascular endothelium.

(2) Robust experimental procedures including kidney perfusion.

(3) Comprehensive set of experiments including appropriate controls.

(4) Disclosure of a new ligand-independent vascular ERα effect.

Weaknesses

(1) Ignoring a possible role of GPER and signaling pathways thereof in the study endpoints is a major flaw in the experimental design.

(2) The regulation of flow-mediated dilation by estrogen has been widely investigated in previous studies.

(3) The gender claim in the title is relatively weak as it is based on just one set of experiments (Figure 1; lines 137-138).

Although the study objective was stated concisely, the Authors generated findings of potential impact in the field that deserve further investigation. A cross-talk between blood flow, NO pathway and membrane-associated ERα appears to emerge from the present work and represents a conceptual advance. However, the role of GPER in this setting deserves to be assessed as well.

Reviewer #4:

Using a variety of genetically-manipulated mouse models, the authors study how membrane associated estrogen receptor alpha (ERα) impacts flow-mediated dilation (FMD). Their collective findings indicate that non-nuclear actions of plasma membrane-associated ERα in endothelial cells support FMD in both sexes via mechanisms that are independent of estrogens. The process likely involves the promotion of NO production and blunting of impact of reactive oxygen species (ROS).

Strengths include the use of multiple genetic manipulations in mice, which allow testing of ERα function as a transcription factor and as a modulator of extra-nuclear signaling initiated by a subpopulation of plasma membrane-associated ERα. The specific requirement for ERα in endothelial cells is also evaluated. FMD is primarily tested in isolated mesenteric resistance arteries, but some key findings are confirmed in uterine arteries. The complementary use of an isolated perfused kidney model is also a strength.

One weakness is the evaluation of levels of ERα expression by quantifying transcript abundance in whole arteries when ERα protein abundance in endothelial cells is of prime importance. In addition, although alterations in NO and ROS are implicated, no insights are gained into how these are impacted by ERα presumably independent of estrogens. Despite the lack of more mechanistic interrogation, the overall observation of an important role for non-nuclear function of plasma membrane-associated ERα in endothelial cells in FMD is important to the field.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your revised manuscript "Membrane estrogen receptor alpha (ERα) optimizes flow-mediated dilation in both sexes, in a ligand-independent manner" for consideration by eLife. Your revised article has been evaluated by a Reviewing Editor and a Senior Editor. the Reviewing Editor has drafted this letter to help you prepare a revised submission.

Essential revisions:

1) The wording "optimization" in the title may be "optimized". The reviewer thinks that e.g. an experimental procedure but not a biological process such as FMD may be optimized. ER participates in or supports FMD.

2) The p values and other statistical parameters could be more appropriately moved from figures to figure legends, especially when asterisks are present.

3) qPCR experiments: "relative expression" may refer to either an housekeeping gene or a reference control sample. Please indicate the first option in figure legends.

4) Figure 5: Western blots should be shown for phospho-eNOS and total eNOS, and for phospho-Akt and total Akt.

5) Line 63-65: The authors state that flow-mediated dilatation depends mainly on NO, citing studies looking at the forearm circulation in humans. This should be stated/clarified. However, in mice (the organism used for experiments in this study) flow-dependent vasodilatation in mesenteric resistance arteries (the vascular bed investigated in this study) is mediated by both, NO and EDH (PMID 16055522; 11282893). Brandes and associates also found that EDH is the main mediator of endothelium-dependent relaxation in murine resistance arteries (PMID 10944233). Moreover, the EDH-shear-dependent response is partly sensitive to non-selective COX-inhibition (PMID 16055522). The authors should discuss these important differences between humans and mice and also should mention the limitation of their study that experiments were conducted in the absence of COX inhibition, and that one cannot fully exclude the involvement of COX-dependent / endothelial-derived prostanoid effects in the effects observed.

6) Lines 112-115: With regard to estrogen effects in men the authors should discuss intracrine, aromatase-mediated production of estradiol which is converted locally in the vasculature from testosterone (PMID 11248122) by which estrogen partly provides protection in male mice from atherosclerosis.

7) Lines 145-147: The authors suddenly introduce compounds targeting GPER, without having made any reference to this receptor in the introduction. It would be helpful if the authors could add a little section to the introduction discussing this receptor as well, also citing a good overview article, such as PMID 21844907

8) Line 144, Line 465-466, : The authors state "the ERα-ERb antagonist and GPER agonist fulvestrant (ICI-182780)." This is partly correct. Fulvestrant is a SERD (selective estrogen receptor downregulator or selective estrogen receptor degrader), which down-regulates/degrades the receptors it targets. This should be corrected.

9) Line 262: findings for the evaluation of changes of gene expression with C451A-ERα are not shown in Figure 7.

10) Lines 335-336: the statement is misleading because chronic effects of E2 (recognizing that the term "chronic" is vague) can be mediated by non-nuclear actions of ERα.

11) Lines 402-403: there is no basis for stating that the decrease in FMD in mice lacking membrane ERα could reflect a feature of premature aging of the endothelium.

12) Figure 9 schematic: there is no evidence that Gai mediates the role of membrane-associated ERα in FMD.

13) P. 21: The reference for G36 provided in the table is not quite correct, the one cited was published to 2010, at a time when G36 had not yet been published. It is suggested to list PMID 27803283 as reference instead.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner" for further consideration by eLife. Your revised article has been evaluated by a Reviewing Editor and a Senior Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1) Previous comment "Lines 145-147: The authors suddenly introduce compounds targeting GPER, without having made any reference to this receptor in the introduction. It would be helpful if the authors could add a little section to the introduction discussing this receptor as well, also citing a good overview article, such as PMID 21844907"

Authors Response: We have added a paragraph in the introduction stating the role of GPER in endothelium-dependent dilation (lines 87-90):

"E2-dependent vasodilation has also been shown to involve the G-protein-coupled estrogen receptor (GPER) in both human and animal arteries [24]. In the rat, GPER activation reduces uterine vascular tone during pregnancy through activation of endothelium-dependent NO production [25]."

It appears that there was a misunderstanding with regard to the previous comment. It was not requested to describe solely the role of GPER in endothelial cell function and NO release. Rather, the authors were expected to expand this section, briefly describing the nature of GPER (namely that it is a 7-transmembrane GPCR located at the endoplasmic reticulum), its natural and synthetic ligands, and its functions (release of NO is just one). Also, the authors should mention that there is both, ligand-dependent and ligand-independent activation (PMID 27803283) of GPER, which should also be discussed in the Discussion section.

2) The "Key Resources Table" in the Methods section includes a number of references:

Hypertension 2007;50:248-54

Hypertension. 2007;50:248-54

EMBO Mol Med. 2014;6:1328-46

Pharmacology 2010: 86, 58- 64

Sci Signal. 2016;9:ra105

J Vasc Res. 2009;46:2 53-64

Am J Physiol 2018;315:H1019-H1026

Cytokine 2009;46,166-70

These references should be added in full to the References section

3) The whole manuscript should be checked for grammar and typos, including "contribute to relaxation of the mesenteric resistance arteries in both male and female through, at least in part, a PI3K-Akt-eNOS pathway [28]." (lines 98-99, page 5) and corrections should be made as needed.

Essential revisions:

1) The ligand-independent activation of membrane ERα is a novel finding. However, simply ignoring a potential role of GPER in such ligand-independent regulation of FMD is a major flaw. The authors should carry out some straight-forward studies to look at the effects of pharmacological inhibition using GPER antagonist, G-36, which turns out to not only inhibit GPER but also via inhibition of constitutive GPER expression, to reduce abundance (and activity) of Nox1. The authors are advised to perform in vitro studies of vessels from WT mice, evaluating dilation in response to flow +/- GPER antagonist, as long as a parallel control study is done evaluating dilation in response to GPER agonist +/- GPER antagonist.

We agree with your comment. Ligand-independent activation of membrane ERα has been previously demonstrated. However, the present finding shows, for the first time its involvement in the endothelium response to flow in resistance arteries.

We performed the experiments requested and added the data to figure 1 (panels I and J) and to the corresponding paragraph in the manuscript (Results section on figure 1 and discussion). We found that G36 incubation did not affect flow-mediated dilation in arteries from WT mice suggesting that GPER may not have a role in this vascular response although it is now clear that GPER has a full place in cardiovascular homeostasis and pathophysiology.

As G36 has an IC50 for G1 and E2 (estradiol) which are very close (165 nM and 112 nM, respectively: Dennis et al., J Steroid Biochem Mol Biol. 2011 Nov;127(3-5):358-66), we tested the effect of G36 on both compounds and observed that G36 inhibited both dilation indued by G1 and E2 (panel J, figure 1).

2) In the Introduction section, the authors are encouraged to cite original works related to non-nuclear signaling by the subpopulation of ER associated with the plasma membrane in endothelium.

We have extended this part of the discussion (Introduction, lines 74 to 93).

3) In the Introduction section, the background information about cardiovascular disease risk in men versus women and the impact of estrogens on risk is not as clear-cut as the authors imply. A more balanced presentation of the clinical evidence is warranted.

We have added more studies describing the protective effect of estrogens with a focus on FMD. This is mainly in the introduction between line 92 and line 111.

4) In the Introduction, a more detailed account of the published literature in the field including studies in humans should be provided The Authors may consider citing some studies from among PMID 19126786; 7642876; 20185791; 26471832; 9176294; 17367797; 28645860; 26734763; 9791075; 16131583; 18319309; 19086257; 9930647.

We have included most of these articles in the introduction and/or in the discussion.

Several articles are from our group and show that flow-mediated outward remodeling involves E2-dependent activation of nuclear function (AF2) of ERα. This remodeling occurs in response to a chronic increase in flow and affects all the layers of the arterial wall. By contrast with FMD which is an acute response involving non-nuclear ERα. We have added a paragraph on this difference to avoid confusion (lines 70-74 and in the discussion: lines 337-349).

5) In the Results section, Page 6, line 106 incorrectly refers the reader to Figure 1B.

We have corrected this error (lines 135-37).

6) In the Results section. Page 6, line 117: fulvestrant is an ERα and ERβ antagonist (pKi = 9 for both) as well as a GPER full agonist (pKi = 7).

We have completed the corresponding text as follows:

“Furthermore, the ERα-ERb antagonist and GPER agonist fulvestrant (ICI-182780) did not alter the FMD” Now in line 144.

Same correction in the Material and Methods section.

7) In the Results section, Figures 2B, 2F and 2J: ERα protein abundance in endothelial cells should be evaluated, even if only feasible from vasculature that would yield more endothelial cells than mesenteric resistance arteries.

We have performed these experiments in endothelial cells isolated from the aorta as too few cells can be obtained from the mesenteric arteries. This new data is shown in Figure 3 B, F and J.

Besides markers of endothelial cells (Tie2 or Tek) and of smooth muscle cells (Cnn1) were quantified in order to control for the quality of the endothelial cells isolation.

Tek and Cnn1 expression levels are in Figure 3, supplement figure 1.

8) In the Results section, Figure 5B: the flow-pressure relationship was shifted left, or up, in C451A-ERα mice versus WT according to the group labeling, and not to the right.

We have corrected this text. Thank you for having noticed this error.

9) In the Results section Page 17, line 281: the use of the term autoregulation here may be confusing, and more specific interpretation would be helpful (and better placed in the Discussion section).

We have modified the sentence as follows:

“Thus, these results suggest that FMD reduction due to the absence of membrane-ERα also affects the capacity of the renal vasculature to produce NO and ATP”

Now in Lines 254 – 257.

10) In the Results section, how does flow increase eNOS Ser1177 phosphorylation and alter H₂O₂ via ERα mechanistically ? Are flow-related changes in Akt phosphorylation in endothelium altered in the absence of ERα?

To respond to these questions, we have added several sets of new data.

We have added new data showing the level of phosphorylation of Akt in AF2⁰ERα, C451A-ERα and R264-ERα mice (and the corresponding WT groups) (Figure 5, panels C, F and I + Figure 5 source data 2 showing all the blots for eNOS, Ph-eNOS, Akt and Ph-Akt).

In addition, we have added new data showing that mito-tempo which reduces ROS production by the mitochondria improved FMD in C451A-ERα mice (figure 7). In figure 7 we also added new data showing that the combination of superoxide dismutase and catalase also improved FMD in arteries from C451A-ERα mice (no effect in WT) and that catalase alone (elimination of H₂O₂ into H2O) reduced FMD in C451A-ERα mice without affecting FMD in WT.

Thus, we may state that ERα reduces mitochondria functioning and its production of ROS and H₂O₂. Indeed, several studies have shown that flow (shear stress) induces a more quiescent and less oxidative phenotype in endothelial cells (i.e.: Doddaballapur et al. Arterioscler Thromb Vasc Biol 2015, 35, (1), 137-45). This reduction in mitochondrial ROS production would thus allow NO to be more efficient (less ROS scavenging NO) and allow a more efficient eNOS activation. Of course, this later issue remains to be further investigated. Nevertheless, an excessive ROS production has been shown to reduce eNOS phosphorylation through increased phosphatase activation (Ding et al. Front Physiol 2020, 11, 566410).

In addition, the effect of catalase on FMD in C451A-ERα mice agrees with previous studies by D. Gutterman’s group showing that H₂O₂ produced by the mitochondria can dilate coronary arteries in response to flow (FMD) in arteries from patients with coronary artery disease. Although this allows keeping some FMD, the dilation remains low (as in C451A-ERα mice in the present study) and in the long term, H₂O₂ remains deleterious.

We have also modified the scheme shown in figure 9 to include these new data.

11) In the Results section, Do non-nuclear actions of plasma membrane-associated ERα influence endothelial cell production of prostaglandins in the setting of FMD?

We have added new data showing that the cyclooxygenase inhibitor indomethacin does not further reduce FMD in the 4 groups of mice studied: ERα-/-, AF2⁰ERα, C451A-ERα and R264-ERα mice (and the corresponding WT groups) (new data added to Figure 4).

In addition, we observed no change in COX1, COX2 and prostacyclin expression level in mesenteric arteries isolated from C451A-ERα mice (Figure 7—figure supplement 2: J, K and L).

EDHF is the third major agent produced by the endothelium in response to flow and it is also involved in the activation of endothelium-dependent dilation by estrogens (reference 31 and 32). Thus, we have also added to the manuscript new data obtained with EETs blocker MSPPOH as EETs are major members of the EDHF family. EETs have been shown to mediate, at least in part, the protective effect of E2 on FMD in hypertensive or old rats (reference 31 and 32, introduction, lines 103-109).

This data (indomethacin and MSPPOH) is discussed lines 360-365.

In Figure 4 we show only L-NNA and indomethacin to avoid overloading the figure.

Figure 4 supplement figure 1 shows the 3 blockers, L-NNA, indomethacin and MSPPOH.

12) In the Results section, do non-nuclear actions of plasma membrane-associated ERα influence mechanosensitive ion channel localization or function in endothelial cells?

Based on the experiments described above, it is most likely that non-nuclear actions of plasma membrane associated ERα involve a reduction in oxidative stress and subsequently a better action of eNOS.

Nevertheless, it is important to decipher the link between flow (shear stress) activation of the extracellular matrix and cell surface and the plasma membrane associated ERα.

As flow has been recently shown to activate Piezo1-dependent release of ATP through pannexin hemi-channels followed by ATP-dependent activation of purinergic receptors which induce NO production by endothelial cells (Wang et al. J Clin Invest 2015, 125, (8), 3077-86; Wang et al., J Clin Invest 2016, 126, (12), 4527-4536), we investigated this pathway in C451A-ERα mice:

First, we found no change in the expression level of Piezo1 and Piezo2 channels in mesenteric arteries isolated from C451A-ERα mice. Similarly, no change in the level of polycystin1 and 2, TRPV4 or integrin α and β was found and no change in purinergic receptors level was found (Figure 7—figure supplement 1: panel G to N, Q, R and S).

Second, we have also performed additional experiments and added the new data to the manuscript showing that the acute response to ATP and to the piezo1 agonist YODA-1 was not affected by the absence of membrane ERα in C451A-ERa mice.

Similarly, the mechanosensitive channel blocker GsMTx4 similarly affected FMD in C451A-ERα and WT mice suggesting that the defect in FMD is probably located down-stream flow sensing. New data added to the discussion, lines 376-381 and shown in Figure 7—figure supplement 3, A, B and C.

As discussed above (point 11), we have also included data obtained with the EETs synthesis blocker MSPPOH. Indeed, as stated above, we used indomethacin to assess the role of prostaglandins in FMD in the mouse models used in the present work. We also used MSPPOH as EETs are produced by the endothelium and activate transient receptor potential (TRP) channel (Campbell WB, Fleming I. Epoxyeicosatrienoic acids and endothelium-dependent responses. Pflugers Arch. 2010 May;459(6):881-95). Nevertheless, this compound did not significantly change FMD after L-NNA and indomethacin blockade suggesting that this pathway and the related channels may not be involved (Figure 4 -, figure supplement 1, shown above in response to point 11).

13) In the Results section, why was the antioxidant TEMPOL specifically selected? Would the Authors expect a similar outcome following treatment with other antioxidants such as e.g. quercetin or dimethyl fumarate?

We used TEMPOL as we have already used it in the past with success. In addition, it is easy to use in the drinking water and mice keep drinking normally.

Nevertheless, we have added new data obtained with mice treated with vitamin C and vitamin E (4 weeks of treatment). This treatment has been shown efficient (Favre J et al. Coronary endothelial dysfunction after cardiomyocyte-specific mineralocorticoid receptor overexpression Am J Physiol Heart Circ Physiol. 2011 Jun;300(6):H2035-43) in reducing ROS.

This new set of experiments shows a similar result than with Tempol with a restoration of FMD in treated C451A-ERα mice treated with the antioxidant cocktail vitamin C and vitamin E. This new data is presented in figure 8 (G to L).

Importantly, we obtained a similar effect with 2 different unrelated antioxidant treatments.

14) In the Results section, does Tempol or PEG-catalase acutely restore FMD ex vivo?

We have added new data showing that the combination of superoxide dismutase and catalase restores FMD in C451A-ERa mice. By contrast, catalase which reduces H₂O₂ level improved FMD in C451A-ERa mice. This result supports the assumption that H₂O₂ may be responsible for the remaining FMD in C451A-ERa mice. This was not observed in WT mice. In addition, the effect of superoxide dismutase + catalase which reduces more globally the production of reactive oxygen species improved FMD in C451A-ERa mice. A similar result was obtained with mito-tempo which reduces mitochondrial ROS. This is discussed in more details above (point 10). New data shown in figure 7.

15) In the Results section, Line 312: Chronic for a 2-week treatment does not sound very appropriate.

We have removed the word chronic and left the duration of the treatment (2 weeks).

Of note, the new antioxidant treatment (vitamin E/vitamin C) added to the revised manuscript was supplied for a longer duration (4 weeks). This is now in figure 8.

16) In the Results section, flow rate may be changed to shear stress in Dyn/cm2: this would help comparing between published works in the topic.

We have added the corresponding shear stress in the Material and Methods and in the figure legends.

17) The Discussion section should be shortened, providing more succinct focused discussion of the interpretation of the findings, their implications, and possible explanations to fill the new knowledge gaps that result from the work.

We shortened the initial discussion. Nevertheless, we also added some text to respond to the comments raised above. Altogether, the discussion remains shorter and hopefully better focused on the results of the present work.

18) In the Conclusion section, Figure 7: the schematic is only in part helpful because how non-nuclear actions of membrane-associated ERα in endothelial cells govern eNOS and ROS in response to flow is not addressed.

We have modified this scheme (now Figure 9) and hope that it now better supports the conclusion of the discussion.

Reviewer #1:

[…] The mechanism of membrane receptor, ligand independent pathway of estrogen receptor alpha that the authors reported lacks novelty as it was already previously described.

We agree that ligand independent involvement of estrogen receptor alpha has been described in other fields, mainly in the field of breast cancer. Nevertheless, its involvement in the acute response to flow of resistance arteries is new and potentially of interest in the field of cardiovascular diseases.

Reviewer #2:

Multiple animal models have been used to test the contribution of membrane estrogen receptors on the vascular dilation induced by flow (flow-mediated dilation, FMD).

The authors propose that the presence of membrane estrogen receptors optimizes flow-mediated dilation. However, the conclusion that ER promotes NO production and inhibits oxidative stress is not fully supported by the data since basal ROS production is not altered in the models without functional ER, and an antioxidant treatment normalizes the dilatory response, and effect that can be independent of a direct NOS activation. The data show that in the absence of functional membrane receptors, flow-mediated dilation is reduced but that it is restored by a treatment with an antioxidant. Thus, these receptors seem not to be necessary for FMD. The involvement of these receptors in FMD remains therefore questionable.

We agree that the link between membrane ERα and eNOS activation by flow remains an open question. To address this question, we have added new data to the manuscript which should help better defining the relation between membrane ERα and the balance between eNOS activity and ROS.

First, we have added data showing that ROS inhibition is also active acutely and restored FMD in vitro in arteries isolated from mice lacking membrane-ERα (C451A-ERα mice) in agreement with the in vivo data obtained previously (initial version of the manuscript) showing that a 2-weeks long treatment with antioxidant TEMPOL restored FMD in C451A-ERα mice (we have also included another treatment with vitamins C and E, 4 weeks). This is also in agreement with our measurement of H₂O₂ in the perfusate of isolated kidneys from C451A-ERα mice. Thus, membrane-ERα is necessary to reduce ROS production, and this allows a better FMD. Therefore, FMD is reduced in the absence of membrane-ERα (no more break on ROS production) and ROS reduction restored FMD. Of course, this could exclude a direct involvement of membrane-ERα in flow-sensing and signal transduction to eNOS as you point out in your comment. To further address this question, we used blockers of mechanosensitive channels (similar effect on C451A-ERα and WT mice) and we tested Yoda1 and ATP-dependent dilation in C451A-ERα and WT mice (no difference).

Thus, it seems that flow activates on one hand the NO pathway and on the other hand flow reduces ROS production through activation of the membrane located ERα. This effect on ROS could involve a reduction in mitochondrial activity as we also found that the inhibition of mitochondrial ROS production with Mito-Tempo restored FMD in C451A-ERα mice (new data added to the manuscript). We have extended the discussion on this mechanism. Of course, the pathway linking flow to membrane-ERα and to the mitochondria remains to be further investigated.

Altogether, these data show that membrane-ERα is involved in the acute response to flow of resistance arteries through a reduction in ROS production.

We have changed the conclusion of the abstract as follows:

“Thus, endothelial membrane ERα promotes NO production through inhibition of oxidative stress and thereby helps to optimize FMD in a ligand-independent manner”

Reviewer #3:

[…] (1) Ignoring a possible role of GPER and signaling pathways thereof in the study endpoints is a major flaw in the experimental design.

We have tested the effect of G36 as suggested. Although G36 did not affect FMD in the present study, GPER is certainly an important player in the control of vascular tone in different conditions. Although GPER does not seem to have a role in FMD in the present work, its role in the pathophysiology of the vascular tree is now well recognized.

(2) The regulation of flow-mediated dilation by estrogen has been widely investigated in previous studies.

We agree that estrogen has major role in restoring FMD in many pathological (cardiovascular and metabolic disorders) or physiological (menopause) conditions. We have added more references on this effect of estrogen in the introduction (lines 96-111).

Nevertheless, the present study does not address the effect of estrogen on FMD. On the contrary, we show in the present study that membrane ERα is involved in FMD independently of its ligand. This effect was observed in healthy young mice, both in females and in males. Importantly, this effect is reminiscent of the first case of ERα gene deficiency in a young man: “The first disruptive mutation in the ERα gene, reported in 1994 in a man who was only 30 years old, was found to be associated with a selective and total absence of FMD. This single yet major clinical observation suggests that ERα-dependent signal transduction could play a role in FMD in males”.

Smith et al. N Engl J Med 1994, 331, (16), 1056-61.

Sudhir et al. Lancet 1997, 349, (9059), 1146-7.

3) The gender claim in the title is relatively weak as it is based on just one set of experiments (Figure 1; lines 137-138)

We agree that most of the experiments were conducted on male mice. Nevertheless, the main effect of the absence of membrane-ERα is a reduction of the amplitude of FMD in both male and female mice. This was observed in mesenteric arteries from intact and ovariectomized mice as well as in the uterine arteries of female mice. This later data was shown in the supplement files, and we moved it to figure 1. Nevertheless, we have removed “male and female mice” from the title.

Although the study objective was stated concisely, the Authors generated findings of potential impact in the field that deserve further investigation. A cross-talk between blood flow, NO pathway and membrane-associated ERα appears to emerge from the present work and represents a conceptual advance. However, the role of GPER in this setting deserves to be assessed as well.

We agree with your comment and have performed the requested experiments. We found that G36 did not affect FMD in the mouse mesenteric artery. We have added the data to figure 1.

Reviewer #4:

[…] One weakness is the evaluation of levels of ERα expression by quantifying transcript abundance in whole arteries when ERα protein abundance in endothelial cells is of prime importance. In addition, although alterations in NO and ROS are implicated, no insights are gained into how these are impacted by ERα presumably independent of estrogens. Despite the lack of more mechanistic interrogation, the overall observation of an important role for non-nuclear function of plasma membrane-associated ERα in endothelial cells in FMD is important to the field.

We have isolated endothelial cells from the aorta to quantify ERα gene expression (data in figure 3). We used the aorta as the quantity of cells that would be obtained from mesenteric resistance arteries would be too small and the isolation techniques used for the aorta does not apply to small resistance arteries. Although more cells are obtained from the aorta, the quantity of cells and consequently the quantity of total tissue and proteins obtained remains very limited. Analysis of the amount of protein in endothelial cells isolated from the mouse vessels is not feasible due to the insufficient amount of biological material combined to the low efficiency of ERα antibody in mice. However, we have adapted the protocol described by Iruela Arispe's team to analyze the expression of ERα in endothelial cells isolated from aorta by Q-PCR (Briot et al., Repression of Sox9 by Jag1 is continuously required to suppress the default chondrogenic fate of vascular smooth muscle cells. Dev Cell. 2014;31(6):707-721).

The results obtained demonstrated that there is no difference in the expression of ERα in the endothelial cells, whatever the genotype of the mice. Besides markers of endothelial cells (Tie2) and of smooth muscle cells (Cnn1) were quantified in order to control for the quality of the endothelial cells isolation. ERα is shown in figure 3. Tie2 (Tek) and Cnn1 are shown in the supplemental figures (Figure 3—figure supplement 1).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

1) The wording "optimization" in the title may be "optimized". The reviewer thinks that e.g. an experimental procedure but not a biological process such as FMD may be optimized. ER participates in or supports FMD.

As recommended, the title is now:

“Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner”.

We have also modified the last sentence of the abstract.

2) The p values and other statistical parameters could be more appropriately moved from figures to figure legends, especially when asterisks are present.

As recommended, p values are now given in the figure legends.

Data and full statistical analysis are also shown in the source data files.

3) qPCR experiments: "relative expression" may refer to either an housekeeping gene or a reference control sample. Please indicate the first option in figure legends.

As recommended, the text of the figure legend (figure 3) is now:

“Esr1 expression level in aortic endothelial cells (expression relative to the housekeeping genes Gapdh, Hprt and Gusb)”

4) Figure 5: Western blots should be shown for phospho-eNOS and total eNOS, and for phospho-Akt and total Akt.

As recommended, Western-blots (selected bands) are now shown in figure 5.

Western blots for all the mice used in the study are shown in the Figure 5 source data 2.

5) Line 63-65: The authors state that flow-mediated dilatation depends mainly on NO, citing studies looking at the forearm circulation in humans. This should be stated/clarified. However, in mice (the organism used for experiments in this study) flow-dependent vasodilatation in mesenteric resistance arteries (the vascular bed investigated in this study) is mediated by both, NO and EDH (PMID 16055522; 11282893). Brandes and associates also found that EDH is the main mediator of endothelium-dependent relaxation in murine resistance arteries (PMID 10944233). Moreover, the EDH-shear-dependent response is partly sensitive to non-selective COX-inhibition (PMID 16055522). The authors should discuss these important differences between humans and mice and also should mention the limitation of their study that experiments were conducted in the absence of COX inhibition, and that one cannot fully exclude the involvement of COX-dependent / endothelial-derived prostanoid effects in the effects observed.

We have modified the text as recommended.

More precisely:

a. Line 63-65: it is now stated that this sentence refers to human studies.

The sentence is now: “FMD measured in the human forearm depends mainly on the acute production of NO…” Line 64 now.

b. In mice FMD in mesenteric resistance arteries is mediated by both, NO and EDH:

We have added a paragraph in the discussion stating the limitations of the study as suggested by your comment. Line 365 to 374. As regard to COX-derivatives, we have added to figure 4 (in response to the previous comments) data obtained with indomethacin. Indeed, the addition of indomethacin to LNNA did not further reduced FMD and acetylcholine-mediated dilation. Similarly, the addition of MSPPOH which block EETs production, did not further reduced FMD and acetylcholine-mediated dilation (supplement figure to Figure 4). Although EETs are major component of the EDHF family, this result suggests that the remaining FMD in the presence of L-NNA, indomethacin and MSPPOH is due to other EDHFs.

6) Lines 112-115: With regard to estrogen effects in men the authors should discuss intracrine, aromatase-mediated production of estradiol which is converted locally in the vasculature from testosterone (PMID 11248122) by which estrogen partly provides protection in male mice from atherosclerosis.

We have added the following sentence in the introduction (now: lines 118-120)

“Of note, testosterone has been shown to reduce early atherogenesis in male mice through its conversion to estrogen by aromatase which is expressed in the arterial wall [42]”.

7) Lines 145-147: The authors suddenly introduce compounds targeting GPER, without having made any reference to this receptor in the introduction. It would be helpful if the authors could add a little section to the introduction discussing this receptor as well, also citing a good overview article, such as PMID 21844907

We have added a paragraph in the introduction stating the role of GPER in endothelium-dependent dilation (lines 87-90):

“E2-dependent vasodilation has also been shown to involve the G-protein-coupled estrogen receptor (GPER) in both human and animal arteries [24]. In the rat, GPER activation reduces uterine vascular tone during pregnancy through activation of endothelium-dependent NO production [25].”

8) Line 144, Line 465-466, : The authors state "the ERα-ERb antagonist and GPER agonist fulvestrant (ICI-182780)." This is partly correct. Fulvestrant is a SERD (selective estrogen receptor downregulator or selective estrogen receptor degrader), which down-regulates/degrades the receptors it targets. This should be corrected.

We have corrected the text: “the estrogen receptor downregulator and GPER agonist fulvestrant (ICI-182780)”. Now: line 149 and line 463

9) Line 262: findings for the evaluation of changes of gene expression with C451A-ERα are not shown in Figure 7.

These findings are shown in the supplement figures attached to Figure 7. This is written in the text as requested in the instruction to the authors: “Figure 7, figure supplement figures 1 and 2” These two supplemental figures were attached to the manuscript (merged files) after the figures (page 68 and 69).

10) Lines 335-336: the statement is misleading because chronic effects of E2 (recognizing that the term "chronic" is vague) can be mediated by non-nuclear actions of ERα.

We removed the sentence “The nuclear functions of ERα are mainly involved in the chronic effects of E2 and ERα activation such as atheroma prevention [7]”

Indeed, you are right, it is a misleading shortcut. In addition, the transition to flow-mediated remodeling is better now.

11) Lines 402-403: there is no basis for stating that the decrease in FMD in mice lacking membrane ERα could reflect a feature of premature aging of the endothelium.

We removed the paragraph. Aging is certainly more complex than a single reduction in membrane-ERα signaling even though it might contribute. A more in-depth investigation is needed before stating this.

12) Figure 9 schematic: there is no evidence that Gai mediates the role of membrane-associated ERα in FMD.

We removed Gai from the scheme.

13) P. 21: The reference for G36 provided in the table is not quite correct, the one cited was published to 2010, at a time when G36 had not yet been published. It is suggested to list PMID 27803283 as reference instead.

We apology for the error. We added the right reference to the table.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1) Previous comment "Lines 145-147: The authors suddenly introduce compounds targeting GPER, without having made any reference to this receptor in the introduction. It would be helpful if the authors could add a little section to the introduction discussing this receptor as well, also citing a good overview article, such as PMID 21844907".

Authors Response: We have added a paragraph in the introduction stating the role of GPER in endothelium-dependent dilation (lines 87-90):

"E2-dependent vasodilation has also been shown to involve the G-protein-coupled estrogen receptor (GPER) in both human and animal arteries [24]. In the rat, GPER activation reduces uterine vascular tone during pregnancy through activation of endothelium-dependent NO production [25]."

It appears that there was a misunderstanding with regard to the previous comment. It was not requested to describe solely the role of GPER in endothelial cell function and NO release. Rather, the authors were expected to expand this section, briefly describing the nature of GPER (namely that it is a 7-transmembrane GPCR located at the endoplasmic reticulum), its natural and synthetic ligands, and its functions (release of NO is just one). Also, the authors should mention that there is both, ligand-dependent and ligand-independent activation (PMID 27803283) of GPER, which should also be discussed in the Discussion section.

We apologize for misunderstanding your previous comment.

We have extended the paragraph on GPER in the introduction (lines 93-104):

“The 7-transmembrane G-protein-coupled estrogen receptor (GPER, formerly known as GPR30) is another receptor located not only at the plasma membrane but also on the membrane of the endoplasmic reticulum that can be activated by E2. […] Thus, both ERα and GPER could contribute to the rapid actions of E2, although their respective roles according to vessel type, species and pathophysiological context remain to be established.”

We have also extended the paragraph on GPER in the discussion including the various physiological functions involving GPER and the various diseases which could benefit from a targeting of GPER (lines 357-366):

“Another membrane receptor for E2 located at the plasma membrane is GPER [27]. Both ligand-dependent and ligand-independent activation of GPER have been reported [61]. […] However, a possible role of unliganded GPER activation cannot be excluded in case of a crosstalk between membrane-dependent Erα and GPER activation.”

2) The "Key Resources Table" in the Methods section includes a number of references:

Hypertension 2007;50:248-54

Hypertension. 2007;50:248-54

EMBO Mol Med. 2014;6:1328-46

Pharmacology 2010: 86, 58- 64

Sci Signal. 2016;9:ra105

J Vasc Res. 2009;46:2 53-64

Am J Physiol 2018;315:H1019-H1026

Cytokine 2009;46,166-70

These references should be added in full to the References section

References are now in full in the References Section.

3) The whole manuscript should be checked for grammar and typos, including "contribute to relaxation of the mesenteric resistance arteries in both male and female through, at least in part, a PI3K-Akt-eNOS pathway [28]." (lines 98-99, page 5) and corrections should be made as needed.

We have rechecked the manuscript.

Author details

1. Julie Favre

   Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France

   Contribution

   Conceptualization, Data curation, Investigation, Methodology, Resources, Validation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Emilie Vessieres

   Competing interests

   No competing interests declared

2. Emilie Vessieres

   1. Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France
   2. CARFI facility, Angers University, Angers, France

   Contribution

   Data curation, Formal analysis, Methodology

   Contributed equally with

   Julie Favre

   Competing interests

   No competing interests declared

3. Anne-Laure Guihot

   1. Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France
   2. CARFI facility, Angers University, Angers, France

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Coralyne Proux

   1. Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France
   2. CARFI facility, Angers University, Angers, France

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Linda Grimaud

   Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France

   Contribution

   Data curation, Methodology, Resources

   Competing interests

   No competing interests declared

6. Jordan Rivron

   1. Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France
   2. CARFI facility, Angers University, Angers, France

   Contribution

   Investigation, Methodology, Validation

   Competing interests

   No competing interests declared

7. Manuela CL Garcia

   1. Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France
   2. CARFI facility, Angers University, Angers, France

   Contribution

   Investigation, Validation

   Competing interests

   No competing interests declared

8. Léa Réthoré

   Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Rana Zahreddine

   INSERM U1297, Paul Sabatier University (Toulouse III) , University Hospital (UHC) of Toulouse, Toulouse, France

   Contribution

   Investigation, Validation, Writing – original draft

   Competing interests

   No competing interests declared

10. Morgane Davezac

    INSERM U1297, Paul Sabatier University (Toulouse III) , University Hospital (UHC) of Toulouse, Toulouse, France

    Contribution

    Data curation, Methodology, Performed experiments requested for the revised manuscript

    Competing interests

    No competing interests declared

11. Chanaelle Fébrissy

    INSERM U1297, Paul Sabatier University (Toulouse III) , University Hospital (UHC) of Toulouse, Toulouse, France

    Contribution

    Investigation, Validation, Visualization

    Competing interests

    No competing interests declared

12. Marine Adlanmerini

    INSERM U1297, Paul Sabatier University (Toulouse III) , University Hospital (UHC) of Toulouse, Toulouse, France

    Contribution

    Conceptualization, Investigation, Validation

    Competing interests

    No competing interests declared

13. Laurent Loufrani

    1. Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France
    2. University Hospital (CHU) of Angers, Angers, France

    Contribution

    Formal analysis, Investigation, Supervision, Validation, Writing – original draft

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3397-2335

14. Vincent Procaccio

    1. Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France
    2. University Hospital (CHU) of Angers, Angers, France

    Contribution

    Methodology, Project administration, Validation

    Competing interests

    No competing interests declared

15. Jean-Michel Foidart

    Groupe Interdisciplinaire de Génoprotéomique Appliquée, Université de Liège, Liège, Belgium

    Contribution

    Methodology, Resources

    Competing interests

    No competing interests declared

16. Gilles Flouriot

    INSERM U1085, IRSET (Institut de Recherche en Santé, Environnement et Travail), University of Rennes, Rennes, France

    Contribution

    Conceptualization, Methodology, Validation

    Competing interests

    No competing interests declared

17. Françoise Lenfant

    INSERM U1297, Paul Sabatier University (Toulouse III) , University Hospital (UHC) of Toulouse, Toulouse, France

    Contribution

    Conceptualization, Methodology, Supervision, Validation, Writing – original draft, Writing – review and editing

    Competing interests

    No competing interests declared

18. Coralie Fontaine

    INSERM U1297, Paul Sabatier University (Toulouse III) , University Hospital (UHC) of Toulouse, Toulouse, France

    Contribution

    Methodology, Validation, Writing – original draft

    Competing interests

    No competing interests declared

19. Jean-François Arnal

    INSERM U1297, Paul Sabatier University (Toulouse III) , University Hospital (UHC) of Toulouse, Toulouse, France

    Contribution

    Conceptualization, Funding acquisition, Writing – original draft

    Competing interests

    No competing interests declared

20. Daniel Henrion

    1. Angers University, MITOVASC, CNRS UMR 6015, INSERM U1083, Angers, France
    2. CARFI facility, Angers University, Angers, France
    3. University Hospital (CHU) of Angers, Angers, France

    Contribution

    Conceptualization, Funding acquisition, Methodology, Project administration, Validation, Writing – original draft

    For correspondence

    daniel.henrion@univ-angers.fr

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-1094-0285

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