obesity has reached pandemic dimensions . in association with insulin resistance , hypertension , and dyslipidaemia , obesity forms white adipose tissue ( wat ) is increased in both obese mice and humans and is characterized by an increase in the inflammatory cytokines and oxidative stress . furthermore , adipocytes can induce aldosterone synthesis in the adrenocortical gland in an endocrine fashion . as a result many of its effects are mediated by activation of its nuclear receptor , the mineralocorticoid receptor ( mr ) . mineralocorticoid receptor antagonists reduce morbidity and mortality in patients with congestive heart failure . in corresponding mouse models , atherosclerosis is initiated at sites of endothelial dysfunction which may be caused by enhanced generation of cox - derived vasoconstricting prostanoids or reactive oxygen species ( ros ) . the increased oxidative stress is mainly due to a disturbed expression of ros - producing enzymes such as nadph oxidase and enzymes involved in antioxidant defense such as glucose-6-phosphate dehydrogenase ( g6pdh ) and glutathione peroxidase ( gpx ) . mr activation induces expression of nadph oxidase subunits , thereby contributing to generation of oxidative stress . furthermore , aldosterone alters expression of prostanoid - producing enzymes ( enhanced ) and g6pdh ( reduced ) that are associated with endothelial dysfunction . mineralocorticoid receptor is expressed in both endothelial cells and adipocytes . while it may induce an array of mediators involved in endothelial dysfunction in the former , it acts in adipocytes as a pro - adipogenic transcription factor . pharmacological mr blockade using eplerenone reduces insulin resistance , macrophage infiltration , expression of pro - inflammatory factors , and ros release in wat . of note , the effects of mr blockade on obesity - induced endothelial dysfunction , the role of the endothelial mr in this context , and the corresponding molecular mediators remain unknown . thus , we assessed endothelial function of lean and diet - induced obese mice , differing in endogenous aldosterone levels , and of lean mice with and without exogenous aldosterone infusion . mineralocorticoid receptor activity was modulated by a pharmacological and a genetic approach . as non - selective receptor binding properties of spironolactone may result in an increase in plasma cortisol and also thereby potentially influence endothelial function , we used in this study , the more selective mr - antagonist eplerenone . to address the role of the endothelial mr , we used a genetic loss - of - function approach targeted to endothelial cells ( tie2-driven endothelial mr deletion ) . online data supplements are available for ( i ) animals , ( ii ) signal transduction and inflammation , ( iii ) blood glucose and components of the renin - angiotensin - aldosterone system , ( iv ) analysis of vascular function , ( v ) isolation of fresh aortic endothelial cells and rna expression analyses , and ( vi ) statistical analyses . to address the contribution of endogenous aldosterone in the development of obesity - induced endothelial dysfunction , we exposed 6-week - old male c57bl/6 mice to a high - fat diet without ( hfd ) or with eplerenone ( hfd epl ) . control mice were fed a normal diet ( nd ) and were considered as lean mice for the purpose of these experiments . to assess a putative activation of the renin aldosterone levels were significantly higher , whereas renin concentration remained unaltered in mice fed an hfd compared with lean mice ( figure  1a and b ) . compared with hfd - fed obese mice , epl treatment of obese mice did not change plasma renin , whereas it increased plasma aldosterone levels ( figure  1a and b ) . feeding an hfd for 14 weeks resulted in a significant increase in body weight and epididymal wat , it increased fasting plasma glucose , and reduced glucose tolerance compared with mice fed an nd . treatment with epl administered with the hfd did not interfere with epididymal wat and total body weight gain . however , epl treatment did prevent worsening of glucose metabolism in these mice ( supplementary material online , table s2 ) . figure  1plasma 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 = 810 ; * 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 = 68 ; * 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 . 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 = 810 ; * 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 = 68 ; * p nd , normal chow diet ; hfd , high - fat diet ; hfd epl , high - fat diet with eplerenone . mrna levels of pro - inflammatory cytokines such as tumour necrosis factor- ( tnf- ) and monocyte chemoattractant protein-1 ( mcp-1 ) tended to be increased ( tnf- ) or were increased ( mcp-1 ) in the epididymal wat of obese mice compared with lean control mice ( figure  1c ) . chronic administration of eplerenone prevented an increase in mrna expression levels of these cytokines as well as macrophage - specific glycoprotein cd68 in the epididymal wat . these data suggest that mr antagonism diminished the obesity - induced pro - inflammatory changes in the epididymal wat . to determine the effects of increased endogenous aldosterone levels in obesity on endothelial function , we performed ex vivo aortic ring contraction / relaxation experiments with aortae from c57bl/6 mice kept on nd , hfd , or hfd epl , respectively ( figure  2 ) . endothelium - dependent relaxation to acetylcholine was blunted in aortae of obese mice starting at a concentration of 3  10 m of acetylcholine and reached a maximal relaxation at 10 m of acetylcholine . endothelium - independent relaxation to sodium nitroprusside ( snp ) was similar in all groups ( figure  2b ) . contractions to kcl and norepinephrine ( ne ) were unaffected in mice fed hfd or hfd epl , respectively ( data not shown ) . 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 = 810 ; * 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 . . 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 = 810 ; * 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 . obesity is known to promote a pro - inflammatory , pro - oxidative , and vasoconstricting state in the vascular endothelium , thereby inducing endothelial dysfunction . to investigate the direct effects of obesity and eplerenone treatment on the genes expressed in aortic endothelial cells , we developed a method to isolate fresh endothelial cells from c57bl/6 mice exposed to nd , hfd , or hfd epl ( supplementary material online , figure s2a and b ) . we observed a slight increase in the mr mrna expression under hfd and an even higher increase upon additional chronic eplerenone treatment ( supplementary material online , figure s3a ) . mineralocorticoid receptor mrna was reduced in endothelial cells of mice in which mr was deleted ( ec mr ) , indicating successful ablation of endothelial mr ( supplementary material online , figure s3b ) . analyses of mediators involved in endothelial dysfunction revealed that prostacyclin synthase and cox-1 expression were upregulated in obesity , whereas cox-2 remained unaltered ( figure  3a ) . eplerenone treatment did neither prevent hfd - induced upregulation of prostacyclin synthase nor cox-1 expression . furthermore , endothelial nitric oxide synthase ( enos ) expression increased in hfd epl compared with nd mice ( figure  3a ) . 
 figure  3obesity - 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 . 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 . given the association of aldosterone with oxidative stress , we tested mrna expression of nadph oxidase subunits in isolated aortic endothelial cells . mrna levels of the p22phox , p47phox , and gp91phox were not affected by diet - induced obesity . however , additional eplerenone treatment decreased the expression of p22phox and p47phox ( figure  3b ) . on the other hand , obesity increased the expression levels of p40phox and rac-1 ; the increase in p40phox was attenuated by eplerenone ( figure  3b ) . mrna expression levels of the antioxidant enzymes sod-1 and sod-3 were lower under hfd compared with nd ( trend for sod-1 ) and hfd epl conditions ; catalase expression remained unaltered ( figure  3c ) . the mrna expression of the ros - scavenging enzyme gpx-1 was enhanced by eplerenone in obesity , whereas gpx-4 and g6pdh remained unchanged ( figure  3d ) . taken together , we observed the generation of an obesity - induced vasoconstrictive and pro - oxidative mrna expression profile in aortic endothelial cells that was partially prevented by pharmacological mr antagonism . to investigate the role of the endothelial mr on obesity - induced metabolic changes , we exposed ec mr mice and their corresponding mr littermates to an nd or hfd for 14 weeks . after 14 weeks on a hfd , both obese mr mice and ec mr showed a similar increase in plasma aldosterone levels ( supplementary material online , figure s4a ) , weight gain , glucose intolerance ( supplementary material online , table s3 ) , as well as expression of pro - inflammatory markers mcp-1 , and cd68 ; tnf- was upregulated only in ec mr mice ( figure  4a ) . figure  4the 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 = 68 ; * p < 0.05 for mr nd vs. mrhfd ; p < 0.05 for ec mr nd vs. ec mrhfd . nd , normal chow diet ; hfd , high - fat diet . 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 = 68 ; * p < 0.05 for mr nd vs. mrhfd ; p < 0.05 for ec mr nd vs. ec mrhfd . nd , normal chow diet ; hfd , high - fat diet . to test the effects of exogenous aldosterone on the endothelial mr , we implanted osmotic minipumps containing aldosterone or vehicle in lean mr and ec mr mice . aldosterone infusion for 2 weeks increased plasma aldosterone to the same extent in both ec mr and mr mice kept on nd ( supplementary material online , figure s4b ) and did not induce the expression of tnf- , mcp-1 , or cd68 in the epididymal wat ( figure  4b ) . these data indicate that endothelial mr deletion neither affects glucose tolerance nor the pro - inflammatory state of the wat of obese or aldosterone - infused lean mice . to determine whether obesity - induced endothelial dysfunction was mediated by the endothelial mr , we exposed ec mr and mr mice to nd or hfd for 14 weeks . compared with lean controls , obese mr mice demonstrated a significant impairment of endothelium - dependent vasodilation , whereas mr deletion in endothelial cells prevented endothelial dysfunction ( figure  5a ) . of note , aortic walls of obese mr mice contained no macrophages ( supplementary material online , figure s5 ) . thus , the endothelial mr mediates both obesity - induced endogenous as well as exogenous aldosterone - induced endothelial dysfunction . figure  5loss 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 = 68 . ( a ) mrand 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 . * 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 ) . 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 = 68 . ( a ) mrand 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 . * 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 ) . cox inhibition using indomethacin normalized endothelial function in aldosterone - infused lean mice to the same extent as endothelial mr deletion ( figure  5c ) . next we evaluated the effects of aldosterone infusion or mr ablation on mediators of inflammation , vasoconstriction , and oxidative stress in aortic endothelial cells . for this purpose , we isolated fresh aortic endothelial cells from lean mr and ec mr mice infused with aldosterone for 2 weeks and determined mrna expression by qpcr . our analyses showed that prostacyclin synthase expression was enhanced by exogenous aldosterone , independent of the presence of endothelial mr ( figure  6a ) ; in contrast , aldosterone - induced cox-1 expression was decreased in ec mr mice , whereas cox-2 and enos expression remained unchanged . figure  6aldosterone - 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 = 68 ; * p < 0.05 . 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 = 68 ; * p < 0.05 . analyses of enzymes enhancing oxidative stress revealed that mrna expression of the nadph oxidase subunit p22phox was upregulated by aldosterone , whereas this effect was abolished in ec mr mice ; gp91phox remained unaltered in all conditions ( figure  6b ) . expression levels of the nadph oxidase subunits p47phox , p40phox , and rac-1 were below the detection limit ( data not shown ) . regarding antioxidant enzymes , expression of sod-1 mrna was decreased by aldosterone , but did not reach statistical significance ( p = 0.077 ) . sod-3 mrna was decreased upon aldosterone infusion and remained low in ec mr mice , both in the presence or absence of aldosterone ( figure  6c ) . thus , aldosterone enhances the expression of mediators involved in the generation of ros and prostanoids in an endothelial mr - dependent manner . to address the contribution of endogenous aldosterone in the development of obesity - induced endothelial dysfunction , we exposed 6-week - old male c57bl/6 mice to a high - fat diet without ( hfd ) or with eplerenone ( hfd epl ) . control mice were fed a normal diet ( nd ) and were considered as lean mice for the purpose of these experiments . to assess a putative activation of the renin aldosterone levels were significantly higher , whereas renin concentration remained unaltered in mice fed an hfd compared with lean mice ( figure  1a and b ) . compared with hfd - fed obese mice , epl treatment of obese mice did not change plasma renin , whereas it increased plasma aldosterone levels ( figure  1a and b ) . feeding an hfd for 14 weeks resulted in a significant increase in body weight and epididymal wat , it increased fasting plasma glucose , and reduced glucose tolerance compared with mice fed an nd . treatment with epl administered with the hfd did not interfere with epididymal wat and total body weight gain . however , epl treatment did prevent worsening of glucose metabolism in these mice ( supplementary material online , table s2 ) . figure  1plasma 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 = 810 ; * 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 = 68 ; * 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 . 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 = 810 ; * 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 = 68 ; * p nd , normal chow diet ; hfd , high - fat diet ; hfd epl , high - fat diet with eplerenone . mrna levels of pro - inflammatory cytokines such as tumour necrosis factor- ( tnf- ) and monocyte chemoattractant protein-1 ( mcp-1 ) tended to be increased ( tnf- ) or were increased ( mcp-1 ) in the epididymal wat of obese mice compared with lean control mice ( figure  1c ) . chronic administration of eplerenone prevented an increase in mrna expression levels of these cytokines as well as macrophage - specific glycoprotein cd68 in the epididymal wat . these data suggest that mr antagonism diminished the obesity - induced pro - inflammatory changes in the epididymal wat . to determine the effects of increased endogenous aldosterone levels in obesity on endothelial function , we performed ex vivo aortic ring contraction / relaxation experiments with aortae from c57bl/6 mice kept on nd , hfd , or hfd epl , respectively ( figure  2 ) . endothelium - dependent relaxation to acetylcholine was blunted in aortae of obese mice starting at a concentration of 3  10 m of acetylcholine and reached a maximal relaxation at 10 m of acetylcholine . endothelium - independent relaxation to sodium nitroprusside ( snp ) was similar in all groups ( figure  2b ) . contractions to kcl and norepinephrine ( ne ) were unaffected in mice fed hfd or hfd epl , respectively ( data not shown ) . 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 = 810 ; * 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 . 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 = 810 ; * 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 . obesity is known to promote a pro - inflammatory , pro - oxidative , and vasoconstricting state in the vascular endothelium , thereby inducing endothelial dysfunction . to investigate the direct effects of obesity and eplerenone treatment on the genes expressed in aortic endothelial cells , we developed a method to isolate fresh endothelial cells from c57bl/6 mice exposed to nd , hfd , or hfd epl ( supplementary material online , figure s2a and b ) . we observed a slight increase in the mr mrna expression under hfd and an even higher increase upon additional chronic eplerenone treatment ( supplementary material online , figure s3a ) . mineralocorticoid receptor mrna was reduced in endothelial cells of mice in which mr was deleted ( ec mr ) , indicating successful ablation of endothelial mr ( supplementary material online , figure s3b ) . analyses of mediators involved in endothelial dysfunction revealed that prostacyclin synthase and cox-1 expression were upregulated in obesity , whereas cox-2 remained unaltered ( figure  3a ) . eplerenone treatment did neither prevent hfd - induced upregulation of prostacyclin synthase nor cox-1 expression . furthermore , endothelial nitric oxide synthase ( enos ) expression increased in hfd epl compared with nd mice ( figure  3a ) . 
 figure  3obesity - 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 . 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 . given the association of aldosterone with oxidative stress , we tested mrna expression of nadph oxidase subunits in isolated aortic endothelial cells . mrna levels of the p22phox , p47phox , and gp91phox were not affected by diet - induced obesity . however , additional eplerenone treatment decreased the expression of p22phox and p47phox ( figure  3b ) . on the other hand , obesity increased the expression levels of p40phox and rac-1 ; the increase in p40phox was attenuated by eplerenone ( figure  3b ) . mrna expression levels of the antioxidant enzymes sod-1 and sod-3 were lower under hfd compared with nd ( trend for sod-1 ) and hfd epl conditions ; catalase expression remained unaltered ( figure  3c ) . the mrna expression of the ros - scavenging enzyme gpx-1 was enhanced by eplerenone in obesity , whereas gpx-4 and g6pdh remained unchanged ( figure  3d ) . taken together , we observed the generation of an obesity - induced vasoconstrictive and pro - oxidative mrna expression profile in aortic endothelial cells that was partially prevented by pharmacological mr antagonism . to investigate the role of the endothelial mr on obesity - induced metabolic changes , we exposed ec mr mice and their corresponding mr littermates to an nd or hfd for 14 weeks . after 14 weeks on a hfd , both obese mr mice and ec mr showed a similar increase in plasma aldosterone levels ( supplementary material online , figure s4a ) , weight gain , glucose intolerance ( supplementary material online , table s3 ) , as well as expression of pro - inflammatory markers mcp-1 , and cd68 ; tnf- was upregulated only in ec mr mice ( figure  4a ) . figure  4the 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 = 68 ; * p < 0.05 for mr nd vs. mrhfd ; p < 0.05 for ec mr nd vs. ec mrhfd . 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 = 68 ; * p < 0.05 for mr nd vs. mrhfd ; p < 0.05 for ec mr nd vs. ec mrhfd . nd , normal chow diet ; hfd , high - fat diet . to test the effects of exogenous aldosterone on the endothelial mr , we implanted osmotic minipumps containing aldosterone or vehicle in lean mr and ec mr mice . aldosterone infusion for 2 weeks increased plasma aldosterone to the same extent in both ec mr and mr mice kept on nd ( supplementary material online , figure s4b ) and did not induce the expression of tnf- , mcp-1 , or cd68 in the epididymal wat ( figure  4b ) . these data indicate that endothelial mr deletion neither affects glucose tolerance nor the pro - inflammatory state of the wat of obese or aldosterone - infused lean mice . to determine whether obesity - induced endothelial dysfunction was mediated by the endothelial mr , we exposed ec mr and mr mice to nd or hfd for 14 weeks . compared with lean controls , obese mr mice demonstrated a significant impairment of endothelium - dependent vasodilation , whereas mr deletion in endothelial cells prevented endothelial dysfunction ( figure  5a ) . of note , aortic walls of obese mr mice contained no macrophages ( supplementary material online , figure s5 ) . infusion of aldosterone for 2 weeks using minipumps blunted endothelial function in lean mice . thus , the endothelial mr mediates both obesity - induced endogenous as well as exogenous aldosterone - induced endothelial dysfunction . figure  5loss 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 = 68 . ( a ) mrand 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 . * 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 ) . 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 = 68 . ( a ) mrand 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 . * 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 ) . cox inhibition using indomethacin normalized endothelial function in aldosterone - infused lean mice to the same extent as endothelial mr deletion ( figure  5c ) . next we evaluated the effects of aldosterone infusion or mr ablation on mediators of inflammation , vasoconstriction , and oxidative stress in aortic endothelial cells . for this purpose , we isolated fresh aortic endothelial cells from lean mr and ec mr mice infused with aldosterone for 2 weeks and determined mrna expression by qpcr . our analyses showed that prostacyclin synthase expression was enhanced by exogenous aldosterone , independent of the presence of endothelial mr ( figure  6a ) ; in contrast , aldosterone - induced cox-1 expression was decreased in ec mr mice , whereas cox-2 and enos expression remained unchanged . figure  6aldosterone - 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 = 68 ; * p < 0.05 . 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 = 68 ; * p < 0.05 . analyses of enzymes enhancing oxidative stress revealed that mrna expression of the nadph oxidase subunit p22phox was upregulated by aldosterone , whereas this effect was abolished in ec mr mice ; gp91phox remained unaltered in all conditions ( figure  6b ) . expression levels of the nadph oxidase subunits p47phox , p40phox , and rac-1 were below the detection limit ( data not shown ) . regarding antioxidant enzymes , expression of sod-1 mrna was decreased by aldosterone , but did not reach statistical significance ( p = 0.077 ) . sod-3 mrna was decreased upon aldosterone infusion and remained low in ec mr mice , both in the presence or absence of aldosterone ( figure  6c ) . thus , aldosterone enhances the expression of mediators involved in the generation of ros and prostanoids in an endothelial mr - dependent manner . this experimental study provides three main novel findings ( figure  7 ) : ( i ) diet - induced obesity generates endothelial dysfunction by enhancing expression of nadph oxidase and through activation of cox-1-dependent pathways in freshly isolated aortic endothelial cells ; ( ii ) genetic deletion of the endothelial mr prevents obesity- as well as aldosterone - induced endothelial dysfunction without affecting glucose tolerance or pro - inflammatory pathways in the wat ; ( iii ) activation of the endothelial mr is sufficient to induce endothelial dysfunction , in part via increased endothelial p22phox expression . 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 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 . our study provides added value in the following contexts : in mice fed an hfd , we observed an increase in plasma aldosterone . this effect was more pronounced if mice were pre - treated with eplerenone ; renin levels remained unaltered . this is in line with publications showing a positive correlation between obesity and plasma aldosterone levels independent of plasma renin activity . since adipocytes induce the release of aldosterone by adrenocortical cells , we propose a renin - independent mechanism in obesity that stimulates aldosterone secretion . we observed a further increase in plasma aldosterone levels upon eplerenone therapy corroborating previous findings . this phenomenon is likely related to a displacement of the mineralocorticoid from its receptor and/or its enhanced production during mr blockade due to a positive physiological feedback loop . we show for the first time that deletion of endothelial mr does not increase plasma aldosterone levels . thus , we propose that the endothelial mr is not involved in the regulatory feedback loop that induces eplerenone - induced aldosterone production . our data reveal that pharmacological mr blockade in diet - induced obesity improved glucose tolerance . in line with our observation , obesity - associated pro - inflammatory changes in wat can contribute to the development of insulin resistance . we and others demonstrated that eplerenone decreases mrna of pro - inflammatory cytokines in wat . moreover , we extend these findings by showing for the first time that endothelial mr ablation neither prevents impaired glucose tolerance nor increases the expression of pro - inflammatory mediators in the obese wat . thus , endothelial mr signalling is neither involved in these obesity - induced diabetic nor pro - inflammatory changes . pharmacological mr antagonism prevented endothelial dysfunction in diet - induced obesity , suggesting that activation of mr with a concomitant release of inflammatory molecules impaired endothelium - dependent relaxation . notably , endothelium - specific mr deletion completely prevented both diet- and aldosterone - induced endothelial dysfunction . these novel findings demonstrate that activation of the endothelial mr is sufficient to mediate endothelial dysfunction in diet - induced obesity . aldosterone increases expression of cox-2 , prostacyclin production , and vascular inflammation . in the context of diet - induced obesity with increased endogenous aldosterone levels our endothelial function studies revealed that pre - treatment of aortic rings with the cox inhibitor indomethacin completely normalized endothelium - dependent relaxations in response to acetylcholine in obese mice . our results corroborate another study reporting that obesity induces endothelial dysfunction in a cox-1-dependent manner . analyses of the prostacyclin synthase revealed that both diet - induced obesity as well as exogenous aldosterone administration enhanced expression of prostacyclin synthase in aortic endothelial cells . in line with our observation , it has been shown that prostacyclin increased endothelial dysfunction in hypertensive rats . furthermore , inhibition of cox that produces prostacyclin synthase substrates , restores aldosterone - induced endothelial dysfunction . of note , neither pharmacological mr antagonism in diet - induced obesity , nor genetic endothelial - specific mr deletion during aldosterone infusion attenuated prostacyclin synthase expression . these findings imply that increased endogenous aldosterone in obesity induces vasoconstricting prostanoids independent of the endothelial mr . however , we consider the corresponding analyses beyond the scope of the current study . we demonstrate that diet - induced obesity was associated with an increased expression of the pro - oxidant nadph subunits ( p40phox and rac-1 ) as well as a reduced expression of the antioxidant proteins sod-1 and -3 . thus , increased ros production may contribute to endothelial dysfunction . indeed , the endothelial mr mediates superoxide generation via rac-1 activation . this is in line with our observation that mr antagonism in obesity conferred beneficial effects on the expression of the nadph oxidase subunits ( p22phox , p47phox , p40phox , and rac-1 ) , antioxidant enzymes sod-1 and -3 , and the ros scavenging enzyme gpx-1 . exogenous aldosterone administration modulated exclusively the nadph oxidase subunit p22phox mrna expression in aortic endothelial cells in an mr - dependent manner . since endothelial mr ablation is sufficient to abolish both obesity- and exogenous aldosterone - induced endothelial dysfunction , we postulate that the endothelial mr - mediated p22phox expression is sufficient to increase a pro - oxidative pathway leading to endothelial dysfunction . overexpression of endothelial mr in mice fed a normal chow induces an enhanced vasoconstrictive response in resistance arteries with a mild hypertension . however , these vessels do not exhibit signs of endothelial dysfunction nor increased oxidative stress . differences in experimental settings ( our study : diet - induced obesity , high cholesterol diet , and constitutive ec mr expression ) are likely to account for the diverse findings . this experimental study provides three main novel findings ( figure  7 ) : ( i ) diet - induced obesity generates endothelial dysfunction by enhancing expression of nadph oxidase and through activation of cox-1-dependent pathways in freshly isolated aortic endothelial cells ; ( ii ) genetic deletion of the endothelial mr prevents obesity- as well as aldosterone - induced endothelial dysfunction without affecting glucose tolerance or pro - inflammatory pathways in the wat ; ( iii ) activation of the endothelial mr is sufficient to induce endothelial dysfunction , in part via increased endothelial p22phox expression . 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 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 . our study provides added value in the following contexts : in mice fed an hfd , we observed an increase in plasma aldosterone . this effect was more pronounced if mice were pre - treated with eplerenone ; renin levels remained unaltered . this is in line with publications showing a positive correlation between obesity and plasma aldosterone levels independent of plasma renin activity . since adipocytes induce the release of aldosterone by adrenocortical cells , we propose a renin - independent mechanism in obesity that stimulates aldosterone secretion . we observed a further increase in plasma aldosterone levels upon eplerenone therapy corroborating previous findings . this phenomenon is likely related to a displacement of the mineralocorticoid from its receptor and/or its enhanced production during mr blockade due to a positive physiological feedback loop . we show for the first time that deletion of endothelial mr does not increase plasma aldosterone levels . thus , we propose that the endothelial mr is not involved in the regulatory feedback loop that induces eplerenone - induced aldosterone production . our data reveal that pharmacological mr blockade in diet - induced obesity improved glucose tolerance . in line with our observation , obesity - associated pro - inflammatory changes in wat can contribute to the development of insulin resistance . we and others demonstrated that eplerenone decreases mrna of pro - inflammatory cytokines in wat . moreover , we extend these findings by showing for the first time that endothelial mr ablation neither prevents impaired glucose tolerance nor increases the expression of pro - inflammatory mediators in the obese wat . thus , endothelial mr signalling is neither involved in these obesity - induced diabetic nor pro - inflammatory changes . pharmacological mr antagonism prevented endothelial dysfunction in diet - induced obesity , suggesting that activation of mr with a concomitant release of inflammatory molecules impaired endothelium - dependent relaxation . notably , endothelium - specific mr deletion completely prevented both diet- and aldosterone - induced endothelial dysfunction . these novel findings demonstrate that activation of the endothelial mr is sufficient to mediate endothelial dysfunction in diet - induced obesity . aldosterone increases expression of cox-2 , prostacyclin production , and vascular inflammation . in the context of diet - induced obesity with increased endogenous aldosterone levels our endothelial function studies revealed that pre - treatment of aortic rings with the cox inhibitor indomethacin completely normalized endothelium - dependent relaxations in response to acetylcholine in obese mice . our results corroborate another study reporting that obesity induces endothelial dysfunction in a cox-1-dependent manner . analyses of the prostacyclin synthase revealed that both diet - induced obesity as well as exogenous aldosterone administration enhanced expression of prostacyclin synthase in aortic endothelial cells . in line with our observation , it has been shown that prostacyclin increased endothelial dysfunction in hypertensive rats . furthermore , inhibition of cox that produces prostacyclin synthase substrates , restores aldosterone - induced endothelial dysfunction . of note , neither pharmacological mr antagonism in diet - induced obesity , nor genetic endothelial - specific mr deletion during aldosterone infusion attenuated prostacyclin synthase expression . these findings imply that increased endogenous aldosterone in obesity induces vasoconstricting prostanoids independent of the endothelial mr . however , we consider the corresponding analyses beyond the scope of the current study . we demonstrate that diet - induced obesity was associated with an increased expression of the pro - oxidant nadph subunits ( p40phox and rac-1 ) as well as a reduced expression of the antioxidant proteins sod-1 and -3 . this is in line with our observation that mr antagonism in obesity conferred beneficial effects on the expression of the nadph oxidase subunits ( p22phox , p47phox , p40phox , and rac-1 ) , antioxidant enzymes sod-1 and -3 , and the ros scavenging enzyme gpx-1 . exogenous aldosterone administration modulated exclusively the nadph oxidase subunit p22phox mrna expression in aortic endothelial cells in an mr - dependent manner . since endothelial mr ablation is sufficient to abolish both obesity- and exogenous aldosterone - induced endothelial dysfunction , we postulate that the endothelial mr - mediated p22phox expression is sufficient to increase a pro - oxidative pathway leading to endothelial dysfunction . overexpression of endothelial mr in mice fed a normal chow induces an enhanced vasoconstrictive response in resistance arteries with a mild hypertension . however , these vessels do not exhibit signs of endothelial dysfunction nor increased oxidative stress . differences in experimental settings ( our study : diet - induced obesity , high cholesterol diet , and constitutive ec mr expression ) are likely to account for the diverse findings . we provide evidence of increased endothelial mr expression in obese compared with lean mice and of a lack of mr expression in endothelial cells of ec mr mice  all at the dna and rna level . despite extensive efforts , we did not succeed in visualizing mr expression at the protein level using various lots of previously described antibodies . since the knockout procedure consistently deletes the gene , we consider that our data provide solid evidence for mr deletion in endothelial cells . as the tie2 promotor used to drive mr deletion in endothelial cells is also active in myeloid cells , we can not conclude that endothelial protection seen in ec mr mice is due exclusively to endothelial mr ablation . moreover , mr ablation in macrophages reduces mr - mediated cardiac pro - inflammatory responses in a mouse model of cardiac fibrosis . since we found no macrophages in the aortae of obese mice with intact mr and we observed similar pro - inflammatory expression patterns in the wat of obese ec mr and mr mice , we propose that tie2-driven ablation of mr in adipose tissue macrophages is unlikely to contribute to endothelial protection in diet - induced obesity in ec mr mice . blood pressure in the different animal groups was not assessed , although a minor increase in blood pressure of obese animals and a decrease in mice lacking the endothelial mr are possible . indeed , increased blood pressure has been observed upon exogenous aldosterone administration and in mice with endothelium - specific mr overexpression . thus , changes in blood pressure could have taken place and may have modified aortic endothelial gene expression and participated in the observed alterations of endothelial function . we demonstrate that both endogenous aldosterone in obesity as well as exogenous aldosterone cause endothelial dysfunction by inducing expression of ros - generating enzymes in an endothelial mr - dependent fashion and by activating the cox-1 pathway independent of the endothelial mr . pharmacological mr blockade abolishes obesity - induced glucose intolerance , adipose tissue inflammation , and endothelial dysfunction . genetic ablation of the mr in endothelial cells identifies the endothelial mr as a crucial mediator of obesity - induced endothelial dysfunction ( figure  7 ) . in addition , changes in blood pressure secondary to endothelial mr ablation and/or the deletion of the mr in macrophages may have contributed to the observed prevention of obesity - induced effects by tie2-driven mr deletion . our experimental findings have the following potential clinical implications : mr - antagonizing drugs appear worth testing for endothelial function in the rapidly growing population of obese patients that are particularly prone to atherosclerosis , hypertension , and insulin resistance . this work was supported by grants from the swiss national science foundation 
 310030 - 130626/1 ( c.m.m . ) , and 3100 - 068118 ( t.f.l . ) the university research priority program integrative human physiology at the university of zurich ( n.s . further support was provided by an unrestricted grant from pfizer ( nyc , usa ) . conflict of interest : c.m.m . received grant from zihp , university of zurich ; msd ; astrazeneca ; elililly ; sirtris ; bayer ; swiss national science foundation ; msd ; astrazeneca ; roche ; has expert testimony to msd ; has patent rights to mabimmune , ch .