Low-Dose Mineralocorticoid Receptor Blockade Prevents Western Diet-Induced Arterial Stiffening in Female Mice

Abstract

Women are especially predisposed to development of arterial stiffening secondary to obesity because of consumption of excessive calories. Enhanced activation of vascular mineralocorticoid receptors impairs insulin signaling, induces oxidative stress, inflammation, and maladaptive immune responses. We tested whether a subpressor dose of mineralocorticoid receptor antagonist, spironolactone (1 mg/kg per day) prevents aortic and femoral artery stiffening in female C57BL/6J mice fed a high-fat/high-sugar western diet (WD) for 4 months (ie, from 4-20 weeks of age). Aortic and femoral artery stiffness were assessed using ultrasound, pressurized vessel preparations, and atomic force microscopy. WD induced weight gain and insulin resistance compared with control diet-fed mice and these abnormalities were unaffected by spironolactone. Blood pressures and heart rates were normal and unaffected by diet or spironolactone. Spironolactone prevented WD-induced stiffening of aorta and femoral artery, as well as endothelial and vascular smooth muscle cells, within aortic explants. Spironolactone prevented WD-induced impaired aortic protein kinase B/endothelial nitric oxide synthase signaling, as well as impaired endothelium-dependent and endothelium-independent vasodilation. Spironolactone ameliorated WD-induced aortic medial thickening and fibrosis and the associated activation of the progrowth extracellular receptor kinase 1/2 pathway. Finally, preservation of normal arterial stiffness with spironolactone in WD-fed mice was associated with attenuated systemic and vascular inflammation and an anti-inflammatory shift in vascular immune cell marker genes. Low-dose spironolactone may represent a novel prevention strategy to attenuate vascular inflammation, oxidative stress, and growth pathway signaling and remodeling to prevent development of arterial stiffening secondary to consumption of a WD.

Keywords: aldosterone; obesity; spironolactone; vascular stiffness.

Conflict of interest statement

Disclosures: The authors have declared that no conflicts of interest exist.

© 2015 American Heart Association, Inc.

Figures

Figure 1

Impaired aortic stiffness and aortic endothelial and smooth muscle cell function in untreated western diet-fed mice (WDC) are improved by MR antagonism (WDSp). (A) Aortic pulse wave velocity (PWV) measured after 2, 3 and 4 months on experimental diets. Values are mean ± SE; N=6–10 per group. (B) Mean (± SE) arterial pressure (n = 3–6/group) and heart rate (n = 6–10/group) at the end of the four month feeding trial did not differ among groups. The micrograph in panel (C) shows the AFM cantilever positioned over the surface of an aortic explant. Panel (D) shows the surface of the extracellular matrix (ECM) after removal of the surrounding adventitia. Panels (E) and (G) show the endothelial cell (EC) and VSMC surfaces, respectively, of intact aortic explants. The bar graphs in panels (F) and (H) demonstrate elevated stiffness in EC and VSMC of WD fed mice and improvement with Sp (n=4–5per group). Vasodilator responses of isolated aortic rings to the endothelium-dependent dilators acetylcholine (I) and insulin (J) and to the endothelium independent vasodilator, sodium nitropruside (SNP) (K). Values are mean ± SE; n=3–6 per group. Control Diet Control (CDC), Control Diet Sp (CDS), Western Diet Control (WDC), and Western Diet Sp (WDSp). Post-hoc comparisons within a time point; *P

Figure 2

Aortic remodeling in untreated western diet-fed mice (WDC) mice is prevented by MR antagonism (WDSp). (A) Representative micrographs show medial wall thickening, peri-aortic fibrosis by (B) VVG and (C) picrosirius red staining and (D) adventitial accumulation of fibronectin. (E) Immunostaining analysis for 3-nitrotyrosine staining. Abbreviations and symbols are the same as in Figure 2 legend. The vessel lumen is indicated by the letter L. Values are mean ± SE; n=5 per group.

Figure 3

Immunoblot analysis of (A) activation of ERK1/2, (B) p-Akt and (C) eNOS expression relative to total Akt and eNOS expression, respectively. Abbreviations and symbols are the same as in Figure 2. Values are mean ± SE; n=3 per group.

Figure 4

Effect of Sp on macrophage polarization. (A) mRNA expression of M1 markers MCP-1 and CD 86, as well as the total macrophage cell marker, CD11b, in aorta. (B) mRNA expression of the M2 marker, IL10, and the ratios of M2/M1 and M2/total macrophage expression in aorta. Values are mean ± SE; n=3 per group; p

Figure 5

Effect of Sp on WD-induced femoral artery endothelial dysfunction and vascular stiffness. Femoral artery constriction to phenylephrine (A) and vasodilation to ACh (B) and sodium nitroprusside (SNP; C), (D) femoral artery strain-stress relationship and (E) femoral artery modulus of elasticity-pressure curve. Femoral artery remodeling is indicated by pressure-diameter relationships, (F), wall/lumen-pressure relationships (G), mean wall thickness-pressure relationship (H) and cross sectional area-pressure relationship (I). Values are mean ± SE; n=3–7 per group; †p