The cholesterol metabolite 27-hydroxycholesterol promotes atherosclerosis via proinflammatory processes mediated by estrogen receptor alpha

Abstract

Oxysterols are cholesterol metabolites that serve multiple functions in lipid metabolism, including as liver X receptor (LXR) ligands. 27-hydroxycholesterol (27HC) is an abundant oxysterol metabolized by CYP7B1. How 27HC impacts vascular health is unknown. We show that elevations in 27HC via cyp7b1 deletion promote atherosclerosis in apoe(-/-) mice without altering lipid status; furthermore, estrogen-related atheroprotection is attenuated. In wild-type mice, leukocyte-endothelial cell adhesion is increased by 27HC via estrogen receptor (ER)-dependent processes. In monocytes/macrophages, 27HC upregulates proinflammatory genes and increases adhesion via ERα. In endothelial cells, 27HC is also proadhesive via ERα, and in contrast to estrogen, which blunts NF-κB activation, 27HC stimulates NF-κB activation via Erk1,2 and JNK-dependent IκBα degradation. Whereas 27HC administration to apoe(-/-) mice increases atherosclerosis, apoe(-/-);erα(-/-) are unaffected. Thus, 27HC promotes atherosclerosis via proinflammatory processes mediated by ERα, and it attenuates estrogen-related atheroprotection. Strategies to lower 27HC may complement approaches targeting cholesterol to prevent vascular disease.

Figure 1

Lipid and 27HC status of wild-type (WT), cyp7b1^−/−, apoe^−/−, and apoe^−/−;cyp7b1^−/−male and female mice. At 12 months of age plasma cholesterol (A) and 27HC concentrations (B) were measured in intact mice. Values are mean±SEM, n=5-10, *p<0.05 vs WT, †p<0.05 vs apoe^−/−. Lipid profiles were also evaluated in samples pooled from 2 male (C) or 2 female mice (D).

Figure 2

27HC increases atherosclerotic lesion formation. Representative images and summary data for aortic root lesions in intact 12 month-old wild-type (WT), cyp7b1^−/−, apoe^−/−, and apoe^−/−;cyp7b1^−/− male and female mice are shown in A and B, respectively. Findings for atherosclerotic lesions in the aortas of male and female mice are provided in C and D, respectively. Summary data are provided in scatter plots. Mean values are indicated by the long horizontal line, and SEM by the short horizontal line. Related data are provided in Table S1. *p<0.05 vs WT, †p<0.05 vs apoe^−/−, Δp<0.05 vs. males. See also Figure S1.

Figure 3

27HC attenuate E₂-related atheroprotection. A. Representative images of aortic roots in female apoe^−/− versus apoe^−/−;cyp7b1^−/− mice ovariectomized at 12 weeks of age, fed a western diet and treated with vehicle, 6 ug/d E₂ or 50 ug/d E₂ for 8 weeks. B. Summary data are provided in scatter plots. Mean values are indicated by the long horizontal line, and SEM by the short horizontal line. *p<0.05 vs apoe^−/−, †p<0.05 vs vehicle. See also Figures S2 and Table S3.

Figure 4

27HC promotes vascular inflammation. A. Macrophage infiltration was compared in the aortas of 12 month-old male apoe^−/− versus apoe^−/−;cyp7b1^−/− mice by quantifying transcript abundance for CD68. B-D. Steady-state mRNA levels for IL-6 (A), MMP-9 (B) and TNF-α (C) normalized to CD68 expression were also compared. In A-D, values are mean±SEM, n=4-6, *p<0.05 vs apoe^−/−. E-I. 27HC promotes leukocyte-endothelial cell adhesion in vivo. Male C57BL/6 mice were treated with vehicle or 27HC, followed by vehicle or TNF-α, and intravital microscopy was then performed to evaluate leukocyte-endothelial cell adhesion in the mesenteric microcirculation. Representative still images are shown for mice treated with vehicle (Veh, E), 27HC (F), TNF-α (G), or TNF-α plus 27HC (H). Summary data are provided in I. Values are mean±SEM, n=7-9, *p<0.05 vs no 27HC, †p<0.05 vs no TNF-α. See also Figure S3, S4A and Movies S1-S4.

Figure 5

27HC has direct proinflammatory actions on monocytes/macrophages and endothelial cells mediated by ERα. A-C. Peritoneal macrophages from wild-type (WT) or erα^−/− mice were treated with vehicle (Veh) or 27HC for 20h, and transcript abundance for TNF-α (A), IL-1β (B) or IL-6 (C) was evaluated. n=3. D-F. Bovine aortic endothelial cells were treated with vehicle or 27HC, and the adhesion of added U937 cells was evaluated. The dose-response to 27HC was assessed (D, n=7), and the involvement of endothelial cell ERα was determined using the selective ERα antagonist MPP (E, n=4) or siRNA knockdown of the receptor (F, n=4). Effective loss of ERα protein with siRNA knockdown is shown in the inset. G. The impact of 27HC on adhesion-promoting mechanisms in monocytes was determined in U937 cells treated 18h with vehicle or 27HC, with or without MPP added, prior to their addition to endothelial cells (n=6). Values are mean±SEM expressed relative to vehicle treatment, *p<0.05 vs vehicle. See also Figure S4B-G.

Figure 6

27HC promotes IκBα degradation and activates NF-κB in endothelial cells via ERα and Erk1,2- and JNK-dependent processes. A. Using immunofluorescence, the subcellular distribution of the NF-kB subunit p65 was evaluated in endothelial cells treated with vehicle, LPS (100 nM) or 27HC (10 uM). p65 shown in green, nuclei stained with Dapi. Values are mean±SEM, n=10-18, *p<0.05 vs vehicle. B-C. The impact of 27HC on IκBα abundance was evaluated in cells treated for 0-120 min with 10 uM 27HC (B), or in cells treated for 30 min with 0-10uM 27HC (C). In B and C, values are mean±SEM, n=6-9 and *p<0.05 vs vehicle. D. The effects of 27HC (10 uM), E₂ (10 nM), or the combination of 27HC and E₂ on IκBα abundance were compared over 30 min. E. The effects of 27HC (10 uM) or LPS (100 ng/ml) on IκBα abundance were compared over 30 min in cells previously transfected with control siRNA or siRNA targeting ERα. F. MAPK activation in response to 27HC (10 uM) or LPS (100 ng/ml) treatment for 0-120 min was evaluated by immunoblot analyses for phosphorylated and total Erk1,2, p38MAPK and JNK. IκBα Ser32 phosphorylation and total protein abundance were also assessed. G. The effect of 27HC (10 uM) on IκBα abundance was compared over 30 min in cells coincubated with vehicle or the MAPK inhibitors PD98059 (1 uM), SB203580 (10 uM), SP600125 (10 uM), or PD98059 plus SP600125. See also Figure S5.

Figure 7

ER mediate the pro-inflammatory and pro-atherosclerotic actions of 27HC in vivo. A. Leukocyte-endothelial adhesion was evaluated by intravital microscopy in wild-type male mice administered vehicle or 27HC without or with cotreatment with ICI 182,780. Values are mean±SEM, n=7-11, *p<0.05 vs no 27HC, †p<0.05 vs no ICI 182,780. B-E. The impact of 27HC administration on atherosclerosis was evaluated in apoe^−/− (B, C) or apoe^−/−;erα^−/− male mice (D,E). Representative aortic root lesions for vehicle or 27HC-treated mice are shown in B and D, and summary data (mean±SEM) are provided in C (n= 12-13) and E (n= 7), *p<0.05 vs vehicle. See also Figure S6.