Estrogen prevent atherosclerosis by attenuating endothelial cell pyroptosis via activation of estrogen receptor α-mediated autophagy

Abstract

Excessive inflammation and the pyroptosis of vascular endothelial cells caused by estrogen deficiency is one cause of atherosclerosis in post-menopausal women. Because autophagy is highly regulated by estrogen, we hypothesized that estrogen can reduce vascular endothelial cell pyroptosis through estrogen receptor alpha (ERα)-mediated activation of autophagy to improve atherosclerosis in post-menopausal stage. Aortic samples from pro-menopausal and post-menopausal women with ascending aortic arteriosclerosis were analyzed, and bilateral ovariectomized (OVX) female ApoE-/- mice and homocysteine (Hcy)-treated HUVECs were used to analyze the effect of estrogen supplementation therapy. The aortic endothelium showed a decrease in ERα expression and autophagy, but presented an increase in inflammation and pyroptosis in female post-menopausal patients. Estrogen treatment accelerated autophagy and ameliorated cell pyroptosis in the cardiac aortas of OVX ApoE-/- mice and Hcy-treated HUVECs. Estrogen had therapeutic effect on atherosclerosis and improved the symptoms associated with lipid metabolism disorders in OVX ApoE-/- mice. Inhibition and silencing of ERα led to a reduction in the autophagy promoting ability of estrogen and aggravated pyroptosis. Moreover, the inhibition of autophagy promoted pyroptosis and abolished the protective effect of estrogen, but had no influence on ERα expression. Thus, the results of the present study demonstrated that post-menopausal women present decreased autophagy and ERα expression and excessive damage to the ascending aorta. In addition, in vitro and in vivo assay results demonstrated that estrogen prevents atherosclerosis by upregulating ERα expression and subsequently induces autophagy to reduce inflammation and pyroptosis.

Keywords: Atherosclerosis; Autophagy; Estrogen; Inflammation; Menopause; Pyroptosis.

Graphical abstract

Fig. 1

Female post-menopausal patients present reduced estrogen receptor levels and autophagy and increased NLRP3 inflammasome and pyroptosis in the ascending aorta. (A) Representative histological images of HE staining in ascending aorta in pre-menopausal women and post-menopausal women. (B) Representative transmission electron microscopy images of ascending aorta in pre-menopausal women and post-menopausal women. The boxed area is showed by a higher magnification. Arrow in red, autophagosome. (C) Representative immunohistochemical staining images of IL-1, IL-18 and Hcy in ascending aorta in pre-menopausal women and post-menopausal women. Up boxed area showed a higher magnification of the intima and the down boxed area showed a higher magnification of the media. (D) Related to C, the intensity analysis of IL-1, IL-18 and Hcy in the endothelium (left panel) and vascular smooth muscle cells (right panel) of ascending aorta in pre-menopausal women and post-menopausal women. In all experiments, n = 12. *P < 0.05, **P < 0.01.

Fig. 2

Estrogen supplementation ameliorates pyroptosis and inflammation in cardiac aorta of OVX ApoE-/- mice and Hcy-treated HUVECs. (A) Representative western blots and relative quantitative analysis of NLRP3, cleaved caspase 1, and GSDMD in cardiac aorta of three groups of mice. (B) Levels of IL-1β in plasma of three groups of mice. (C) Levels of IL-18 in the plasma of three groups of mice. (D) Levels of Hcy in plasma of three groups of mice. (E) Representative western blots and relative quantitative analysis of NLRP3, cleaved caspase 1, and GSDMD in HVUECs cells. (F) Representative western blots and relative quantitative analysis of IL-1 and IL-18 in HVUECs cells. In all experiments, n = 6. **P < 0.01.

Fig. 3

Estrogen supplementation accelerates autophagy in the cardiac aortas of OVX ApoE-/- mice and Hcy-treated HUVECs. (A) Representative western blots and relative quantitative analysis of Beclin1, LC3B, and SQSTM1 in cardiac aorta in mice of three groups. (B) Representative western blots and relative quantitative analysis of Beclin1, LC3B, and SQSTM1 in HVUECs cells. (C) After transfection of HUVECs with GFP-LC3 adenovirus, immunofluorescence images of cells were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂. DAPI was used to stain the nucleus. Mean optical density analysis of GFP was performed. Scale bar: 50 μm. In all experiments, n = 6. **P < 0.01.

Fig. 4

Estrogen supplementation ameliorates atherosclerosis in post-menopausal mice via autophagy induction. (A) Representative transmission electron microscopy images of cardiac aorta in mice of three groups. The boxed area is showed by a higher magnification. Arrow in red, autophagosome. (B) Representative images of oil red O staining of cardiac aorta in mice of three groups and quantitative analysis of the percentage of plaque area. (C) Representative images of oil red O staining of sections of aortic root in mice of three groups and quantitative analysis of the percentage of oil red O positive staining area. Scale bar: 50 μm. (D) Levels of TC, TG, HDL-c and LDL-c in plasma in mice of three groups. In all experiments, n = 6. **P < 0.01.

Fig. 5

Estrogen supplementation induces autophagy via estrogen receptor α. (A) Representative western blots and relative quantitative analysis of ERα and ERβ in cardiac aorta of three groups of mice. (B) Representative western blots and relative quantitative analysis of ERα and ERβ in HVUECs cells. (C) Representative western blots and relative quantitative analysis of ERα and ERβ with or without 12 h of application of 60 nM Hcy to HUVECs in the presence or absence of 10 nM E₂ or/and 10 μM MPP. (D) Representative western blots and relative quantitative analysis of ERα and ERβ with or without 12 h of application of 60 nM Hcy to HUVECs in the presence or absence of 10 nM E₂ or/and 10 μM PHTPP. (E) After transfection of HUVECs with GFP-LC3 adenovirus, immunofluorescence images of cells were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂ or/and 10 μM MPP. DAPI was used to stain the nucleus. Mean optical density analysis of GFP was performed. Scale bar: 50 μm. (F) After transfection of HUVECs with GFP-LC3 adenovirus, immunofluorescence images of cells were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂ or/and 10 μM PHTPP. DAPI was used to stain the nucleus. Mean optical density analysis of GFP was performed. Scale bar: 50 μm. In all experiments, n = 6. **P < 0.01.

Fig. 6

Estrogen supplementation ameliorates cell pyroptosis by upregulating autophagy via estrogen receptor α. (A) Representative western blots and relative quantitative analysis of Beclin1, LC3B, and SQSTM1 in HVUECs cells that transfected with Control siRNA or ERα siRNA. (B) Representative western blots and relative quantitative analysis of NLRP3, cleaved caspase 1, and GSDMD in HVUECs cells that transfected with Control siRNA or ERα siRNA. (C) Representative western blots and relative quantitative analysis of IL-1β and IL-18 in HVUECs cells that transfected with Control siRNA or ERα siRNA. (D) Immunofluorescence images of cells that transfected with Control siRNA or ERα siRNA were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂. Green: GSDMD, red: ERα. DAPI was used to stain the nucleus. Mean optical density analysis of GSDMD and ERα was performed. Scale bar: 50 μm. (E) Immunofluorescence images of cells that transfected with Control siRNA or ERα siRNA were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂. Red: Caspase-1. DAPI was used to stain the nucleus. Mean optical density analysis of Caspase-1 was performed. Scale bar: 50 μm. In all experiments, n = 6. **P < 0.01.

Fig. 7

Inhibition of autophagy abrogates the protective effect of estrogen supplementation on pyroptosis without negatively affecting ERα expression. (A) Representative western blots and relative quantitative analysis of Beclin1, LC3B, and SQSTM1 with or without 12 h of application of 60 nM Hcy to HUVECs in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. (B) Representative western blots and relative quantitative analysis of NLRP3, cleaved caspase 1, and GSDMD with or without 12 h of application of 60 nM Hcy to HUVECs in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. (C) Representative western blots and relative quantitative analysis of IL-1β and IL-18 with or without 12 h of application of 60 nM Hcy to HUVECs in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. (D) After transfection of HUVECs with GFP-LC3 adenovirus, immunofluorescence images of cells were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. DAPI was used to stain the nucleus. Mean optical density analysis of GFP was performed. Scale bar: 50 μm. (E) Immunofluorescence images of cells were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. Green: GSDMD, red: ERα. DAPI was used to stain the nucleus. Mean optical density analysis of GSDMD and ERα was performed. Scale bar: 50 μm. (F) Immunofluorescence images of cells were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E₂ or/and 10 μM 3-MA. Red: Caspase-1. DAPI was used to stain the nucleus. Mean optical density analysis of Caspase-1 was performed. Scale bar: 50 μm. In all experiments, n = 6. **P < 0.01.