Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity

Abstract

Aims: Aldosterone plays a crucial role in cardiovascular disease. 'Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the 'endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis.

Methods and results: C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high 'endogenous' aldosterone) and in 'exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR.

Conclusion: Obesity-induced endothelial dysfunction depends on the 'endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.

Keywords: Aldosterone; Endothelial; Mineralocorticoid receptor; Obesity.

Figure 1

Plasma aldosterone is increased in obesity and further elevated by mineralocorticoid receptor antagonism; obesity-induced expression of pro-inflammatory cytokines in white adipose tissue is attenuated by mineralocorticoid receptor antagonism. C57BL/6 mice on ND, HFD, and HFD EPL were analysed concerning (A) plasma-renin and (B) plasma-aldosterone levels; n = 8–10; *P < 0.05. (C) mRNA levels of epididymal fat pad in C57BL/6 mice on ND, HFD, and HFD EPL, normalized to ND levels; n = 6–8; *P < 0.05 compared with ND levels, ^#P < 0.05 compared with HFD EPL. ND, normal chow diet; HFD, high-fat diet; HFD EPL, high-fat diet with eplerenone.

Figure 2

Mineralocorticoid receptor antagonism prevents obesity-induced worsening of endothelial function. Endothelial function studies in C57BL/6 mice on ND, HFD, and HFD EPL. Responses of aortic rings to increasing doses of (A) acetylcholine (Ach), mediating endothelial release of endogenous nitric oxide, and (B) sodium nitroprusside (SNP), a nitric oxide donor. See Supplementary material online, Table S4 for EC50 and Emax values; n = 8–10; *P < 0.05 for ND vs. HFD; ^#P < 0.05 for HFD vs. HFD EPL. ND, normal chow diet; HFD, high-fat diet; HFD EPL, high-fat diet with eplerenone.

Figure 3

Obesity-induced pro-inflammatory and pro-oxidative changes are modulated by mineralocorticoid receptor antagonism in aortic endothelial cells. Aortic endothelial cell mRNA levels in C57BL/6 mice on ND, HFD, HFD EPL after 14 weeks (A) prostacyclin synthase, COX-1, COX-2, eNOS, (B) p22phox, 47phox, gp91phox, p40phox, Rac-1, (C) SOD-1, SOD-3, catalase, (D) G6PDH, GPx-1, GPx-4, normalized to ND levels, n = 10, *P < 0.05, ^#P < 0.01. ND, normal chow diet; HFD, high-fat diet; HFD EPL, high-fat diet with eplerenone.

Figure 4

The obesity-induced increase in the expression of pro-inflammatory cytokines in white adipose tissue is independent of endothelial mineralocorticoid receptor; aldosterone infusion in lean animals does not induce inflammation. mRNA levels of the epididymal fat pad in wild-type (MR^+/+) and endothelial-specific MR knockout (EC MR^−/−) mice (A) on ND or HFD and (B) vehicle- or aldosterone-infused, standardized to S12 and normalized to ND levels; n = 6–8; *P < 0.05 for MR^+/+ ND vs. MR^+/+HFD; ^#P < 0.05 for EC MR^−/− ND vs. EC MR^−/−HFD. ND, normal chow diet; HFD, high-fat diet.

Figure 5

Loss of endothelial MR prevents obesity-induced endothelial dysfunction and COX inhibition restores aldosterone-induced endothelial dysfunction. The vascular response of aortic rings pre-constricted with norepinephrine to increasing doses of the endothelium-dependent vasodilator acetylcholine (Ach) was measured in wild-type (MR^+/+) and endothelial-specific MR knockout (EC MR^−/−) mice; n = 6–8. (A) MR^+/+and EC MR^−/− mice on ND and HFD; *P < 0.05 for MR^+/+ ND vs. MR^+/+ HFD; ^#P < 0.05 for MR^+/+ HFD vs. EC MR^−/− HFD; ^$P < 0.05 for MR^+/+ HFD vs. EC MR^−/− ND. (B) MR^+/+ and EC MR^−/− mice after aldosterone or vehicle infusion; *P < 0.05 for MR^+/+ vehicle vs. MR^+/+ aldo; ^#P < 0.05 for MR^+/+ aldo vs. EC MR^−/− aldo; ^$P < 0.05 for MR^+/+ aldo vs. EC MR^−/− vehicle. (C) MR^+/+ on ND and HFD or after aldosterone or vehicle infusion; rings were pre-treated for 30 min with indomethacin. See Supplementary material online, Table S4 for EC50 and Emax values. *P < 0.05 for MR^+/+ aldo vs. MR^+/+ aldo (indomethacin-treated); ^#P < 0.05 for MR^+/+ aldo vs. EC MR^−/− aldo; ^$P < 0.05 for MR^+/+ aldo vs. EC MR^−/− aldo (indomethacin-treated). ND, normal chow diet; HFD, high-fat diet.

Figure 6

Aldosterone-induced expression of COX-1 and reactive oxygen species-generating enzymes in endothelial aortic cells depend on endothelial mineralocorticoid receptor. Aortic endothelial cell mRNA levels in wild-type (MR^+/+) and endothelial-specific MR knockout (EC MR^−/−) mice after aldosterone or vehicle infusion (A) prostacyclin synthase, COX-1, COX-2, eNOS, (B) p22phox, gp91phox, (C) SOD-1, SOD-3, (D) G6PDH, GPx-1, GPx-4, standardized to S12 and normalized to ND levels; n = 6–8; *P < 0.05. ND, normal chow diet.

Figure 7

Mechanisms of aldosterone-induced endothelial dysfunction in obesity. The expression of NADPH oxidase subunit p22phox can be blocked by both eplerenone and endothelial mineralocorticoid receptor ablation. Therefore, p22phox seems to be the crucial mediator in inducing aldosterone-induced endothelial dysfunction in aortic endothelial cells of obese mice through genomic (solid arrow) or non-genomic (dashed arrow) effects.