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. Author manuscript; available in PMC: 2022 Jun 18.
Published in final edited form as: Biochem Biophys Res Commun. 2021 Apr 22;558:29–35. doi: 10.1016/j.bbrc.2021.04.019

Estrogen activates endothelial exocytosis

Christine Kim 1, Kyungmoo Yea 1, Craig N Morrell 2, Youngtae Jeong 1,†, Charles J Lowenstein 3
PMCID: PMC8149209  NIHMSID: NIHMS1697477  PMID: 33895548

Abstract

Estrogen replacement for post-menopausal women has been proven beneficial and widely used in treating post-menopausal symptoms such as hot flush, vaginal dryness and osteoporosis. However, its cardiovascular benefit is still controversial. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17β-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT), but not the ERβ agonist diarylpropionitrile (DPN), induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 minutes and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or p38 inhibitor, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data indicate that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway, leading to vascular thromboembolism. Our data further suggest that potential cardiovascular side effects should be considered for post-menopausal E2 supplement.

Keywords: Estrogen, exocytosis, endothelial cells, platelet, thromboembolism

1. Introduction

17β-estradiol (E2) is the predominant form of estrogen in females during the reproductive years. E2 is about 10- to 100-fold more potent than other forms of estrogen such as estrone or estriol [1]. As such, E2 is recommended to prevent or treat post-menopausal symptoms such as hot flush, vagal dryness and osteoporosis, as a combined form with progesterone.

However, the cardiovascular benefit of E2 is still under controversy. Earlier basic studies accumulated reports on the beneficial role of post-menopausal E2 supplement in the cardiovascular system. For example, E2 prevented the apoptosis of endothelial cells and endothelial progenitor cells [2,3]. Also, E2 enhanced NO production and subsequent vascular relaxation [4,5]. However, large-scale clinical trials, including the Women’s Health Initiative (WHI) and the Heart and Estrogen/Progestin Replacement Study (HERS), showed an unexpected result that the group with estrogen replacement therapy rather increased cardiovascular risks, such as coronary events and venous thromboembolism [6–8]. Despite potential flaws in the design of those studies [9–11], subsequent observational studies and large-scale meta-analyses have undisputedly demonstrated the cardiovascular side effect of a high dose of oral estrogen [12–17]. Nonetheless, a body of basic studies indicated the positive side of E2 in the heart and blood vessels [18–20]. Therefore, more comprehensive studies about the effect of E2 are needed to resolve this discrepancy between clinical and basic research data.

As a steroid hormone, the action of E2 is mediated by its two nuclear receptors, ERα and ERβ. After binding of E2 to ERα or ERβ, the ligand-receptor complex exerts genomic effects by translocating into the nucleus and promoting gene transcription. Additionally, E2 also makes rapid actions without inducing new gene transcription. Since the non-genomic actions of E2 include triggering signaling cascades, the membrane-bound receptor was thought to mediate non-genomic actions of E2 [21,22]. However, recent studies revealed that ERα and ERβ, which are located in cytoplasm, also mediate non-genomic actions of E2 [23,24].

Endothelial exocytosis of WPBs is one of the key steps leading to vascular inflammation and thrombosis [25,26]. WPB is an endothelial granule containing numerous pro-inflammatory and pro-thrombotic mediators, including vWF, IL-8 and P-selectin. Upon stimulation by diverse vascular stimuli such as thrombin and histamine, WPBs are rapidly exocytosed from endothelial cells, contributing to leukocyte and platelet adhesion to endothelial cells and promoting vascular inflammation and thrombosis.

In our previous study, we demonstrated that aldosterone, a steroid hormone regulating Na+ and K+ homeostasis and blood volume, triggers endothelial exocytosis [27]. In order to show that aldosterone-induced exocytosis is not the general characteristic of steroid hormones, we found that estradiol also triggers endothelial exocytosis, while progesterone and hydrocortisone do not. We therefore set out to further investigate the role and underlying mechanism of estrogen in endothelial exocytosis as follows.

2. MATERIALS AND METHODS

2.1. Reagents

Aldosterone, 17β-estradiol, Spironolactone, and Mifepristone, propyl pyrazole triol (PPT), diarylpropionitrile (DPN), fulvestrant (ICI-182,780), histamine, U0126, and SB203580 were purchased from Sigma. Thrombin was purchased from Enzyme Research Laboratories. BCECF-AM was purchased from Thermo Fisher.

2.2. Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza-Clonetics. HUVECs were cultured in EGM-2 media supplemented with 2% serum and growth factors included in a kit (Bullet Kit; Lonza-Clonetics) under 37°C, 5% CO2. Endothelial cells were purchased at passage 3 and used within passage 6.

2.3. Endothelial exocytosis experiment

Endothelial exocytosis was performed as previously described [28]. In brief, HUVECs, free of mycoplasma, were washed and placed in EGM-2 media without growth factors and serum. After the stimulation with various drugs during defined time points at 37°C, the supernatant from each well was collected separately. The concentration of vWF and IL-8 released into the media was measured by an ELISA (American Diagnostica).

2.4. siRNA knockdown of Estrogen Receptor

Endothelial cells were electroporated with siRNA against ERα or a control siRNA (Dharmacon) using a nucleofector (Amaxa). Electroporated cells were incubated for 48h at 37°C and stimulated with E2.

2.5. Platelet adhesion assay

Normal healthy blood donors without aspirin or a nonsteroidal anti-inflammatory agent use within 10 days before the blood draw were recruited, and informed consent was obtained from all subjects. Human blood collection was approved by the Institutional Review Board at the Johns Hopkins University Hospital. All methods were carried out in accordance with relevant guidelines and regulations. Whole blood was collected by venipuncture into sodium citrate anticoagulant tubes and centrifuged at 180 × g for 15 min to isolate the top layer of platelet-rich plasma. Platelets were incubated with BCEF-AM for 1 h at 4°C. Prior to co-culture with platelets, HUVECs were pre-treated with E2 or other reagents at 37°C and then, BCEF-loaded platelets were incubated for 15 min at 4°C. The culture was washed with HBSS three times and imaged by a digital fluorescence microscope.

2.6. Statistical Analysis

Statistical analyses were performed using student t-test. Data are presented as mean ± standard error of the mean (S.E.M.) of 3 or more independent experiments. P < 0.05 was considered significant.

3. Results

3.1. Estrogen activates endothelial exocytosis of Weibel-Palade Bodies

One of the unexpected findings from large-scale clinical trials including the WHI and the HERS is that estrogen supplement for post-menopausal women is highly associated with coronary events or vascular thromboembolism. Such observations suggest that estrogen could elicit vascular inflammation and thrombosis [6–8]. Also, in our previous study, E2 as well as aldosterone promoted vWF release from endothelial cells, while progesterone and hydrocortisone did not [27]. We therefore aimed to test whether estrogen activates endothelial exocytosis. Upon the treatment, E2 dose-dependently elicited vWF release from HUVECs, starting from 10 nM, which is close to physiological estrogen concentration (Fig. 1A). E2 also induces the release of IL-8, another component of WPBs in endothelial cells, also starting from 10 nM (Fig. 1B). These data show that E2 stimulates endothelial exocytosis of WPBs.

Figure 1.

Figure 1.

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Estrogen activates endothelial exocytosis. HUVECs were treated with 17β-estradiol (E2) for 1 hr and the release of VWF or IL-8 was measured by ELISA. (A) Dose response for vWF release. Increasing concentrations of E2 cause a dose dependent increase in endothelial release of vWF (N=4). (B) Dose response for IL-8. Increasing concentrations of E2 cause an increase in endothelial release of IL-8 (N=3). All data from A-B are presented as mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

3.2. Estrogen activates endothelial exocytosis through ERα

We first investigated whether estrogen-induced endothelial exocytosis is mediated through estrogen’s nuclear receptors. To this end, we employed ICI-182,780 (Fulvestrant), which binds to estrogen’s nuclear receptors and degrades them. Although E2 induced endothelial exocytosis from 10 nM, we selected 100 nM E2 concentration for mechanistic studies in order to make a clear interfering effect. ICI-182,780 treatment dose-dependently interfered with estrogen-induced endothelial exocytosis, while it did not interfere with histamine-induced endothelial exocytosis (Fig. 2A–B). Also, Mifepristone, a progesterone antagonist, and Spironolactone, an aldosterone antagonist, did not abolish estrogen-induced endothelial exocytosis (Fig. 2C). These data suggest that estrogen-induced endothelial exocytosis is mediated through classical nuclear estrogen receptors.

Figure 2.

Figure 2.

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Estrogen activates endothelial exocytosis through the ERα receptor. HUVECs were treated with E2, inhibitors or agonists of estrogen signaling and the release of vWF was measured by ELISA. (A-B) The estrogen receptor inhibitor ICI 182,780 (ICI, 100 nM) was pre-treated 1 hr before E2 (100 nM) treatment (A) or histamine treatment (B) and vWF release was measured. (C) 100 nM of mifepristone (M) or spironolactone (S) was pre-treated 1 hr before E2 treatment, and vWF release was measured. (D) HUVECs were treated with the ERα agonist, PPT, or the ERβ agonist, DPN for 1 hr, and vWF release was measured. (E-F) ERα was silenced 2 days before E2 treatment (E), and vWF release was measured (F). All data from A-F were repeated three times and are presented as mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant).

We next investigated which estrogen’s nuclear receptors - ERα and ERβ - mediates the estrogen-induced endothelial exocytosis. When we treated HUVECs with the ERα agonist PPT or the ERβ agonist DPN, only PPT elicited vWF release (Fig. 2D). Consistently, the silencing of ERα interfered with the E2-induced vWF release (Fig. 2E–F). Taken together, these data show that ERα mediates estrogen-induced endothelial exocytosis.

3.3. Estrogen-induced exocytosis is mediated non-genomically through the MAP kinase pathway

Although the classical action of nuclear receptors is promoting gene transcription upon ligand binding, they also employ pre-existing proteins as their downstream mediators. In order to distinguish whether E2-induced endothelial exocytosis is mediated through genomic or non-genomic action, we first measured the kinetics of vWF release following E2 treatment. As shown in Fig. 3A, E2 treatment stimulated vWF release as early as 15 minutes, which is too quick to rely on new gene transcription. To further support this finding that E2 non-genomically activates endothelial exocytosis, we pre-treated HUVECs with actinomycin D, a well-known transcription inhibitor, or cycloheximide, a well-known translation inhibitor, prior to E2 treatment. Neither actinomycin D nor cycloheximide pre-treatment interfered with E2-induced vWF release (Fig. 3B–C).

Figure 3.

Figure 3.

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Estrogen activates endothelial exocytosis through rapid, non-genomic pathways. HUVECs were treated with E2 and inhibitors of transcription, translation or MAPK signaling, and the release of VWF was measured by ELISA. (A) Time course study: E2 (100 nM) causes endothelial cells to release VWF over time (N=4). (B) Actinomycin D (2.5 μM) was pre-treated for 2 hr before E2 treatment (N=3). (C) Cyclohexamide (1 μM) was pre-treated for 6 hr before E2 treatment (N=3). (D) U0126 or SB203580 (10 μM) was pre-treated for 1 hr before E2 treatment (N=4). All data from A-D are presented as mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant).

We further investigated the downstream signaling pathways mediating E2-induced endothelial exocytosis. Among the intracellular signaling pathways, the MAP kinase signaling pathway is one of the most rapidly responding pathways to diverse stimuli. We therefore tested whether the MAP kinase signaling pathway mediates E2-induced endothelial exocytosis. Pre-treatment of either ERK inhibitor (U0126) or p38 inhibitor (SB203580) interfered with E2-induced vWF release (Fig. 3D). Taken together, E2 non-genomically induced endothelial exocytosis through the MAP kinase signaling pathway.

3.4. Estrogen treatment enhances in vitro platelet adhesion to endothelial cells

When WPBs are exocytosed, vWF and IL-8 are released while P-selectin is externalized. The released vWF mediates adhesion and aggregation of platelets, while P-selectin functions as an adhesion molecule for platelets and inflammatory cells to endothelial cells. Thus, we hypothesized that E2 treatment enhances platelet adhesion to endothelial cells. To test our hypothesis, we pre-treated HUVEC with a vehicle, E2, or histamine for 1 h before the addition of BCEF-loaded platelets. After incubating at 4°C for 15 min, unbound platelets were washed, and the co-cultured cells were imaged with a digital camera. E2 and histamine treatment led to a comparable outcount of the number of platelets bound to HUVECs (Fig. 4A–B).

Fig 4.

Fig 4.

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Estrogen treatment enhances in vitro platelet adhesion to endothelial cells. (A-B) HUVECs were pre-treated with ICI-182,780 for 1 hr and then treated with E2 or histamine for 1 hr. Then, BCEF-AM loaded platelets were co-cultured with HUVECs at 4 °C for 15 min and imaged with a digital fluorescent camera. Representative figure (A) and bar graph (B) of the number of platelets adhered to HUVECs were shown. (C-D) HUVECs were pre-treated with U0126 for 1 hr before E2 treatment. BCEF-AM loaded platelets were co-cultured with HUVECs at 4 °C for 15 min and imaged with a digital fluorescent camera. Representative figure (C) and bar graph (D) of the number of platelets adhered to HUVECs were shown. All data from A-D were repeated four times and are presented as mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001). All scale bars, 10 μm.

In Fig. 2A and 3C, we have shown that E2-induced endothelial exocytosis is mediated non-genomically through the ERα-MAP kinase signaling pathway. We therefore examined whether inhibition of this pathway interferes with platelet adherence to endothelial cells. ER inhibition by ICI-182,780 pre-treatment interfered with E2-induced platelet binding to endothelial cells (Fig. 4A–B). Also, the pre-treatment of U0126, an ERK inhibitor, interfered E2-induced platelet binding to endothelial cells (Fig. 4C–D).

Taken together, these data indicate that E2-induced endothelial exocytosis promotes the interaction between platelets and endothelial cells, leading to vascular thromboembolism.

4. DISCUSSION

In this study, we demonstrate that E2, the active form of estrogen, activates endothelial exocytosis, promoting platelet adhesion to endothelial cells. E2-induced endothelial exocytosis is mediated by the non-genomic ERα-MAPK signaling. We further demonstrated that estrogen-induced WPB exocytosis enhances platelet adhesion to endothelial cells. Considering the controversies about the benefits and mechanisms of estrogen replacement therapy, we believe that our findings have important implications regarding these unsettling issues.

Early studies, including the WHI and the HERS, discovered the role of estrogen in vascular thrombosis and inflammation. Notwithstanding potential flaws of study design, subsequent observational studies and large meta-analyses have clearly established the association between estrogen and cardiovascular events when a high dose of estrogen is orally administered [12–17]. However, basic studies still report contradicting results. Nonetheless, it appears that the role of estrogen in thromboembolism is quite well supported by experimental studies, in line with clinical data. Prior studies demonstrated that estrogen increased thrombin formation [29,30]. Our data also indicate that estrogen can increase the risk of thromboembolism by enhancing platelet adhesion to vascular endothelial cells.

In contrast, the role of estrogen in cardioprotection or vascular inflammation is still unclear. It appears that in vitro studies show its cardiovascular benefits, including prevention of endothelial apoptosis [2,3], attenuation of monocyte adhesion to endothelial cells [19], and inhibition of LPS-induced monocyte activation [31,32]. However, in vivo studies reported pro-inflammatory responses of macrophages by chronic administration of estrogen [33,34], implicating that treatment duration and mode of experimental modeling can confound the real effect. Also, it is possible that estrogen differentially influences different immune cell populations. For example, E2 selectively increased T lymphocyte population during the early stage of vascular injury while decreased monocyte / macrophages and granulocyte populations [35]. In these regards, our study’s merit lies in the demonstration of E2-induced endothelial exocytosis, which leads to both pro-inflammatory and pro-thrombotic consequences.

Interestingly, a study from decades ago reported similar observations as ours; vWF release from endothelial cells by estrogen [36]. While they simply measured vWF levels 2 days after estrogen treatment, we further demonstrated that vWF release by estrogen is through very rapid WPB exocytosis. The classical action of steroid hormones is mediated through intracellular steroid hormone receptors, which enter into the nucleus after binding with their ligands and promote gene transcription. Therefore, it is thought that these rapid actions of steroid hormones take alternative pathway utilizing pre-existing signaling cascades other than the genomic route. Although membrane-coupled receptors were postulated and identified to mediate the non-genomic action of steroid hormones [21,22], a body of literature has also shown that classical steroid hormone receptors can could elicit signaling cascades [23,24]. Our data add to the growing body of literatures describing rapid, non-genomic effects of estrogen, particularly in endothelial cells [37–40]. One notable study employed a triple point mutant version of ERα, which is defective in rapid signaling but still competent in transcription regulation. Another interesting study showed that ERα mutation abrogates the rapid signaling of E2 while causing no difference in gene expression [41]. In this study, they showed that estrogen non-genomically regulates the migration and proliferation of endothelial cells and, more importantly, monocyte adhesion to endothelial cells [39]. We also demonstrate here that E2 induced endothelial exocytosis very rapidly (Figure 1C), which is mediated through the ERα-MAPK pathway. Considering that WPB exocytosis accompanies P-selectin exposure, it is possible that E2-induced, rapid, endothelial exocytosis increased monocyte adhesion to endothelial cells, which was reported by the prior study.

In conclusion, we demonstrate that E2 increases the platelet adhesion to endothelial cells through endothelial exocytosis, supporting the correlation between increased levels of E2 and the risk of thrombosis. Although most literature comes to the same conclusion that estrogen contributes to vascular thrombosis and inflammation, other studies are still reporting the anti-inflammatory effect of estrogen, suggesting an opportunity for the fine-tuned usage of estrogen. The current hormone replacement therapy regimen is pro-thrombotic and pro-inflammatory in post-menopausal women, but subtle changes in the dose, duration, or combination of estrogen could be anti-inflammatory and still relieve post-menopausal symptoms. Additional work is required to fully understand the action and mechanisms of estrogen in various situations and clearly resolve this issue.

Highlights.

  • Estrogen induced endothelial-exocytosis

  • Estrogen-induced endothelial exocytosis is a very rapid non-genomic action

  • Estrogen-induced endothelial exocytosis is mediated through ERα-MAP kinase signaling pathway

  • Estrogen-induced endothelial exocytosis promotes platelet adhesion to endothelial cells, potentially contributing to vascular thromboembolism

Acknowledgments

Grant Support

This work was supported by grants from American Heart Association (AHA, 0815093E; Y. Jeong), NIH NHLBI R01 HL134894 7 and R33 HL141791 04 (C.J.L.), and R01 HL124018 (C.N.M.). Y. Jeong was also supported by the DGIST Start-Up Fund Program (2018080012) and the DGIST R&D Program (20-CoE-BT-04).

Footnotes

Conflicts of interest: None to declare

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References

  • [1].Trachsler A, Thorn GW, Labhart A, Bürgi H, Dodsworth-Phillips J, Constam GR, Courvoisier B, Fischer JA, Froesch ER, Grob P, Clinical Endocrinology: Theory and Practice, Springer Berlin Heidelberg; 2012. [Google Scholar]
  • [2].Alvarez RJ, Gips SJ, Moldovan N, Wilhide CC, Milliken EE, Hoang AT, Hruban RH, Silverman HS, Dang CV, Goldschmidt-Clermont PJ, 17beta-estradiol inhibits apoptosis of endothelial cells, Biochem Biophys Res Commun 237 (1997) 372–381. 10.1006/bbrc.1997.7085. [DOI] [PubMed] [Google Scholar]
  • [3].Strehlow K, Werner N, Berweiler J, Link A, Dirnagl U, Priller J, Laufs K, Ghaeni L, Milosevic M, Bohm M, Nickenig G, Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation, Circulation 107 (2003) 3059–3065. 10.1161/01.CIR.0000077911.81151.30. [DOI] [PubMed] [Google Scholar]
  • [4].Mendelsohn ME, Nongenomic ER -mediated activation of endothelial nitric oxide synthase: how does it work? What does it mean?, Circ Res 87 (2000) 956–960. 10.1161/01.res.87.11.956. [DOI] [PubMed] [Google Scholar]
  • [5].Schulz E, Anter E, Zou MH, Keaney JF Jr., Estradiol-mediated endothelial nitric oxide synthase association with heat shock protein 90 requires adenosine monophosphate-dependent protein kinase, Circulation 111 (2005) 3473–3480. 10.1161/CIRCULATIONAHA.105.546812. [DOI] [PubMed] [Google Scholar]
  • [6].Grady D, Herrington D, Bittner V, Blumenthal R, Davidson M, Hlatky M, Hsia J, Hulley S, Herd A, Khan S, Newby LK, Waters D, Vittinghoff E, Wenger N, Group HR, Cardiovascular disease outcomes during 6.8 years of hormone therapy: Heart and Estrogen/progestin Replacement Study follow-up (HERS II), JAMA 288 (2002) 49–57. 10.1001/jama.288.1.49. [DOI] [PubMed] [Google Scholar]
  • [7].Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E, Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group, JAMA 280 (1998) 605–613. 10.1001/jama.280.7.605. [DOI] [PubMed] [Google Scholar]
  • [8].Manson JE, Hsia J, Johnson KC, Rossouw JE, Assaf AR, Lasser NL, Trevisan M, Black HR, Heckbert SR, Detrano R, Strickland OL, Wong ND, Crouse JR, Stein E, Cushman M, I. Women’s Health Initiative, Estrogen plus progestin and the risk of coronary heart disease, N Engl J Med 349 (2003) 523–534. 10.1056/NEJMoa030808. [DOI] [PubMed] [Google Scholar]
  • [9].Grodstein F, Clarkson TB, Manson JE, Understanding the divergent data on postmenopausal hormone therapy, N Engl J Med 348 (2003) 645–650. 10.1056/NEJMsb022365. [DOI] [PubMed] [Google Scholar]
  • [10].Hodis HN, Mack WJ, Henderson VW, Shoupe D, Budoff MJ, Hwang-Levine J, Li Y, Feng M, Dustin L, Kono N, Stanczyk FZ, Selzer RH, Azen SP, Group ER, Vascular Effects of Early versus Late Postmenopausal Treatment with Estradiol, N Engl J Med 374 (2016) 1221–1231. 10.1056/NEJMoa1505241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Prentice RL, Manson JE, Langer RD, Anderson GL, Pettinger M, Jackson RD, Johnson KC, Kuller LH, Lane DS, Wactawski-Wende J, Brzyski R, Allison M, Ockene J, Sarto G, Rossouw JE, Benefits and risks of postmenopausal hormone therapy when it is initiated soon after menopause, Am J Epidemiol 170 (2009) 12–23. 10.1093/aje/kwp115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Canonico M, Oger E, Plu-Bureau G, Conard J, Meyer G, Levesque H, Trillot N, Barrellier MT, Wahl D, Emmerich J, Scarabin PY, Estrogen G. Thromboembolism Risk Study, Hormone therapy and venous thromboembolism among postmenopausal women: impact of the route of estrogen administration and progestogens: the ESTHER study, Circulation 115 (2007) 840–845. 10.1161/CIRCULATIONAHA.106.642280. [DOI] [PubMed] [Google Scholar]
  • [13].Laliberte F, Dea K, Duh MS, Kahler KH, Rolli M, Lefebvre P, Does the route of administration for estrogen hormone therapy impact the risk of venous thromboembolism? Estradiol transdermal system versus oral estrogen-only hormone therapy, Menopause 25 (2018) 1297–1305. 10.1097/GME.0000000000001232. [DOI] [PubMed] [Google Scholar]
  • [14].Lidegaard O, Lokkegaard E, Svendsen AL, Agger C, Hormonal contraception and risk of venous thromboembolism: national follow-up study, BMJ 339 (2009) b2890. 10.1136/bmj.b2890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Oedingen C, Scholz S, Razum O, Systematic review and meta-analysis of the association of combined oral contraceptives on the risk of venous thromboembolism: The role of the progestogen type and estrogen dose, Thromb Res 165 (2018) 68–78. 10.1016/j.thromres.2018.03.005. [DOI] [PubMed] [Google Scholar]
  • [16].Scarabin PY, Oger E, Plu-Bureau G, Estrogen THRS Group, Differential association of oral and transdermal oestrogen-replacement therapy with venous thromboembolism risk, Lancet 362 (2003) 428–432. 10.1016/S0140-6736(03)14066-4. [DOI] [PubMed] [Google Scholar]
  • [17].van Hylckama Vlieg A, Helmerhorst FM, Vandenbroucke JP, Doggen CJ, Rosendaal FR, The venous thrombotic risk of oral contraceptives, effects of oestrogen dose and progestogen type: results of the MEGA case-control study, BMJ 339 (2009) b2921. 10.1136/bmj.b2921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Fatima LA, Campello RS, Santos RS, Freitas HS, Frank AP, Machado UF, Clegg DJ, Estrogen receptor 1 (ESR1) regulates VEGFA in adipose tissue, Sci Rep 7 (2017) 16716. 10.1038/s41598-017-16686-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Gao H, Liang M, Bergdahl A, Hamren A, Lindholm MW, Dahlman-Wright K, Nilsson BO, Estrogen attenuates vascular expression of inflammation associated genes and adhesion of monocytes to endothelial cells, Inflamm Res 55 (2006) 349–353. 10.1007/s00011-006-5194-z. [DOI] [PubMed] [Google Scholar]
  • [20].Maggioli E, McArthur S, Mauro C, Kieswich J, Kusters DH, Reutelingsperger CP, Yaqoob M, Solito E, Estrogen protects the blood-brain barrier from inflammation-induced disruption and increased lymphocyte trafficking, Brain Behav Immun 51 (2016) 212–222. 10.1016/j.bbi.2015.08.020. [DOI] [PubMed] [Google Scholar]
  • [21].Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr., Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF, Mol Endocrinol 14 (2000) 1649–1660. 10.1210/mend.14.10.0532. [DOI] [PubMed] [Google Scholar]
  • [22].Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER, A transmembrane intracellular estrogen receptor mediates rapid cell signaling, Science 307 (2005) 1625–1630. 10.1126/science.1106943. [DOI] [PubMed] [Google Scholar]
  • [23].Wang M, Wang Y, Weil B, Abarbanell A, Herrmann J, Tan J, Kelly M, Meldrum DR, Estrogen receptor beta mediates increased activation of PI3K/Akt signaling and improved myocardial function in female hearts following acute ischemia, Am J Physiol Regul Integr Comp Physiol 296 (2009) R972–978. 10.1152/ajpregu.00045.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Wu Q, Chambliss K, Umetani M, Mineo C, Shaul PW, Non-nuclear estrogen receptor signaling in the endothelium, J Biol Chem 286 (2011) 14737–14743. 10.1074/jbc.R110.191791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Lowenstein CJ, Morrell CN, Yamakuchi M, Regulation of Weibel-Palade body exocytosis, Trends Cardiovasc Med 15 (2005) 302–308. 10.1016/j.tcm.2005.09.005. [DOI] [PubMed] [Google Scholar]
  • [26].Schillemans M, Karampini E, Kat M, Bierings R, Exocytosis of Weibel-Palade bodies: how to unpack a vascular emergency kit, J Thromb Haemost 17 (2019) 6–18. 10.1111/jth.14322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Jeong Y, Chaupin DF, Matsushita K, Yamakuchi M, Cameron SJ, Morrell CN, Lowenstein CJ, Aldosterone activates endothelial exocytosis, Proc Natl Acad Sci U S A 106 (2009) 3782–3787. 10.1073/pnas.0804037106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Matsushita K, Morrell CN, Cambien B, Yang SX, Yamakuchi M, Bao C, Hara MR, Quick RA, Cao W, O’Rourke B, Lowenstein JM, Pevsner J, Wagner DD, Lowenstein CJ, Nitric oxide regulates exocytosis by S-nitrosylation of N-ethylmaleimide-sensitive factor, Cell 115 (2003) 139–150. 10.1016/s0092-8674(03)00803-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Ohashi R, Sugimura M, Kanayama N, Estrogen administration enhances thrombin generation in rats, Thromb Res 112 (2003) 325–328. 10.1016/j.thromres.2003.11.014. [DOI] [PubMed] [Google Scholar]
  • [30].Wessler S, Estrogen-associated thromboembolism, Ann Epidemiol 2 (1992) 439–443. 10.1016/1047-2797(92)90093-6. [DOI] [PubMed] [Google Scholar]
  • [31].Pelekanou V, Kampa M, Kiagiadaki F, Deli A, Theodoropoulos P, Agrogiannis G, Patsouris E, Tsapis A, Castanas E, Notas G, Estrogen anti-inflammatory activity on human monocytes is mediated through cross-talk between estrogen receptor ERalpha36 and GPR30/GPER1, J Leukoc Biol 99 (2016) 333–347. 10.1189/jlb.3A0914-430RR. [DOI] [PubMed] [Google Scholar]
  • [32].Vegeto E, Ghisletti S, Meda C, Etteri S, Belcredito S, Maggi A, Regulation of the lipopolysaccharide signal transduction pathway by 17beta-estradiol in macrophage cells, J Steroid Biochem Mol Biol 91 (2004) 59–66. 10.1016/j.jsbmb.2004.02.004. [DOI] [PubMed] [Google Scholar]
  • [33].Calippe B, Douin-Echinard V, Delpy L, Laffargue M, Lelu K, Krust A, Pipy B, Bayard F, Arnal JF, Guery JC, Gourdy P, 17Beta-estradiol promotes TLR4-triggered proinflammatory mediator production through direct estrogen receptor alpha signaling in macrophages in vivo, J Immunol 185 (2010) 1169–1176. 10.4049/jimmunol.0902383. [DOI] [PubMed] [Google Scholar]
  • [34].Calippe B, Douin-Echinard V, Laffargue M, Laurell H, Rana-Poussine V, Pipy B, Guery JC, Bayard F, Arnal JF, Gourdy P, Chronic estradiol administration in vivo promotes the proinflammatory response of macrophages to TLR4 activation: involvement of the phosphatidylinositol 3-kinase pathway, J Immunol 180 (2008) 7980–7988. 10.4049/jimmunol.180.12.7980. [DOI] [PubMed] [Google Scholar]
  • [35].Xing D, Miller A, Novak L, Rocha R, Chen YF, Oparil S, Estradiol and progestins differentially modulate leukocyte infiltration after vascular injury, Circulation 109 (2004) 234–241. 10.1161/01.CIR.0000105700.95607.49. [DOI] [PubMed] [Google Scholar]
  • [36].Harrison RL, McKee PA, Estrogen stimulates von Willebrand factor production by cultured endothelial cells, Blood 63 (1984) 657–664. [PubMed] [Google Scholar]
  • [37].Edgar AR, Judith PY, Elisa DS, Rafael CR, Glucocorticoids and estrogens modulate the NF-kappaB pathway differently in the micro- and macrovasculature, Med Hypotheses 81 (2013) 1078–1082. 10.1016/j.mehy.2013.10.007. [DOI] [PubMed] [Google Scholar]
  • [38].Hohmann N, Xia N, Steinkamp-Fenske K, Forstermann U, Li H, Estrogen Receptor Signaling and the PI3K/Akt Pathway Are Involved in Betulinic Acid-Induced eNOS Activation, Molecules 21 (2016). 10.3390/molecules21080973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Lu Q, Schnitzler GR, Ueda K, Iyer LK, Diomede OI, Andrade T, Karas RH, ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells, PLoS One 11 (2016) e0152807. 10.1371/journal.pone.0152807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Suman M, Giacomello M, Corain L, Ballarin C, Montelli S, Cozzi B, Peruffo A, Estradiol effects on intracellular Ca(2+) homeostasis in bovine brain-derived endothelial cells, Cell Tissue Res 350 (2012) 109–118. 10.1007/s00441-012-1460-2. [DOI] [PubMed] [Google Scholar]
  • [41].Adlanmerini M, Febrissy C, Zahreddine R, Vessieres E, Buscato M, Solinhac R, Favre J, Anquetil T, Guihot AL, Boudou F, Raymond-Letron I, Chambon P, Gourdy P, Ohlsson C, Laurell H, Fontaine C, Metivier R, Le Romancer M, Henrion D, Arnal JF, Lenfant F, Mutation of Arginine 264 on ERalpha (Estrogen Receptor Alpha) Selectively Abrogates the Rapid Signaling of Estradiol in the Endothelium Without Altering Fertility, Arterioscler Thromb Vasc Biol 40 (2020) 2143–2158. 10.1161/ATVBAHA.120.314159. [DOI] [PubMed] [Google Scholar]

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