Author response: Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner

Estrogen receptor alpha (ERα) activation by estrogens prevents atheroma through its nuclear action, whereas plasma membranelocated ERα accelerates endothelial healing. The genetic deficiency of ERα was associated with a reduction in flowmediated dilation (FMD) in one man. Here, we evaluated ex vivo the role of ERα on FMD of resistance arteries. FMD, but not agonist (acetylcholine, insulin)mediated dilation, was reduced in male and female mice lacking ERα (Esr1-/mice) compared to wildtype mice and was not dependent on the presence of estrogens. In C451AERα mice lacking membrane ERα, not in mice lacking AF2dependent nuclear ERα actions, FMD was reduced, and restored by antioxidant treatments. Compared to wildtype mice, isolated perfused kidneys of C451AERα mice revealed a decreased flowmediated nitrate production and an increased H2O2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligandindependent manner. Editor's evaluation Using multiple genetically modified mouse models, the authors have demonstrated a novel role of membrane associated estrogen receptor alpha (ERα) signaling to modulate flowmediated dilation (FMD) in a ligandindependent manner. Specifically, the results indicate that nonnuclear actions of membrane estrogen receptor α in endothelial cells support flowmediated vasodilatation in animals of both sexes via mechanisms that are independent of estrogenic ligands, involving NO production and an attenuation of the NOinactivating effects of reactive oxygen species. These findings highlight a novel role of ligandindependent activation of membrane estrogen receptor α in regulation of vascular physiology and possibly in disease, adding to the recently introduced paradigm shift in the understanding of estrogen and estrogen receptor function. RESEARCH ARTICLE *For correspondence: daniel. henrion@ univangers. fr These authors contributed equally to this work Competing interest: The authors declare that no competing interests exist. Funding: See page 21 Received: 23 March 2021 Accepted: 26 November 2021 Published: 29 November 2021 Reviewing Editor: Noriaki Emoto, Kobe Pharmaceutical University, Japan Copyright Favre et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Research article Cell Biology Favre, Vessieres, et al. eLife 2021;10:e68695. DOI: https:// doi. org/ 10. 7554/ eLife. 68695 2 of 28 Introduction 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 cardioand cerebrovascular 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 pressureinduced smooth muscle contraction and flowmediated 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 (BillonGalés et al., 2009) and neointimal proliferation (Smirnova et al., 2015), and accelerate reendothelialization of injured arteries (Brouchet et al., 2001). Numerous actions of 17betaestradiol (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 nonnuclear 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 ERK12, 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 Ca within minutes (LantinHermoso et al., 1997). By contrast, in HUVECs, E2 induces a rapid production of NO and cGMP independent of an increase in intracellular Ca (CaulinGlaser et al., 1997). This rapid nongenomic activation of eNOS involves Akt/PKB (Florian et al., 2004) and MAP kinasedependent mechanisms (Chen et al., 1999). Estradiolinduced endotheliumindependent 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 membraneassociated ERα, we demonstrated that the acute vasodilator effect of E2 and its accelerative effect on reendothelialization are mediated by membraneassociated ERα (Adlanmerini et al., 2014; Zahreddine et al., 2021). On the other hand, E2 exerts protective effects against atheroma, angiotensin 2induced hypertension, and neointimal hyperplasia through its nuclear effects (Guivarc’h et al., 2018). The 7transmembrane Gproteincoupled 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 GPERselective agonists and antagonists with the use of GPERknockout 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 G1 reduces uterine vascular tone during pregnancy through activation of endotheliumdependent NO production (Tropea et al., 2015). Likewise, the G1induced relaxation of the mesenteric resistance arteries in both male and female rats is mainly mediated by the PI3KAkteNOS pathway (Peixoto et al., 2017). Noteworthy, GPER partly contributes to E2dependent 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, endotheliumdependent dilation of subcutaneous arteries is reduced in postmenopausal women compared to premenopausal women (Kublickiene Research article Cell Biology Favre, Vessieres, et al. eLife 2021;10:e68695. DOI: https:// doi. org/ 10. 7554/ eLife. 68695 3 of 28 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 endotheliumderived 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 E2mediated 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 adrenergicdependent 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 activatio