Endothelial Mineralocorticoid Receptor Mediates Diet-Induced Aortic Stiffness in Females

Abstract

Rationale: Enhanced activation of the mineralocorticoid receptors (MRs) in cardiovascular tissues increases oxidative stress, maladaptive immune responses, and inflammation with associated functional vascular abnormalities. We previously demonstrated that consumption of a Western diet (WD) for 16 weeks results in aortic stiffening, and that these abnormalities were prevented by systemic MR blockade in female mice. However, the cell-specific role of endothelial cell MR (ECMR) in these maladaptive vascular effects has not been explored.

Objective: We hypothesized that specific deletion of the ECMR would prevent WD-induced increases in endothelial sodium channel activation, reductions in bioavailable nitric oxide, increased vascular remodeling, and associated increases in vascular stiffness in females.

Methods and results: Four-week-old female ECMR knockout and wild-type mice were fed either mouse chow or WD for 16 weeks. WD feeding resulted in aortic stiffness and endothelial dysfunction as determined in vivo by pulse wave velocity and ex vivo by atomic force microscopy, and wire and pressure myography. The WD-induced aortic stiffness was associated with enhanced endothelial sodium channel activation, attenuated endothelial nitric oxide synthase activation, increased oxidative stress, a proinflammatory immune response and fibrosis. Conversely, cell-specific ECMR deficiency prevented WD-induced aortic fibrosis and stiffness in conjunction with reductions in endothelial sodium channel activation, oxidative stress and macrophage proinflammatory polarization, restoration of endothelial nitric oxide synthase activation.

Conclusions: Increased ECMR signaling associated with consumption of a WD plays a key role in endothelial sodium channel activation, reduced nitric oxide production, oxidative stress, and inflammation that lead to aortic remodeling and stiffness in female mice.

Keywords: Western diet; inflammation; macrophages; nitric oxide; vascular stiffness.

Figure 1. WD induced EC stiffness and aortic relaxation dysfunction is prevented in ECMR^−/− female mice

(A) Aortic pulse wave velocity (PWV) measured in vivo after 16 week feeding trial on WD. Values are mean± SE; n=6 to 10 per group. (B) The ex vivo measurement of endothelium stiffness by using atomic force microscopy. n=4-5 per group. (C) A representative deflection image in EC shows clear stress fibers in the cytoskeleton. ECMR^−/− prevented aldosterone treatment (10⁻⁸ M) - induced cultured EC stiffness in vitro. Values are mean±SE; n=6 per group. *P<0.05 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.

Figure 2. ECMR mediates ENaC expression in WD-induced aortic relaxation dysfunction. WD ECMR^+/+ increased MR expression

(A), which bind the hormone response element (nGnACAnnnTGTnCn) on the site of ENaC promoter (B) and prompted an increase in ENaC expression (C). ECMR^−/− prevented WD (C) and Aldosterone (D)-induced expression of ENaC in vivo and in vitro, respectively. Representative images immunostaining for ENaC (E) and SGK1 (F) in ECs with corresponding measures of average gray scale intensities. Scale bar = 50 μm. n=4 to 5 per group. *P<0.01 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.

Figure 3. In vivo amiloride administration ameliorated WD-impaired aortic relaxation and flow-induced mesenteric artery dilation in ECMR^+/+ female mice

Vasodilator responses of isolated aortic rings to the endothelium-dependent dilators, acetylcholine (A) and to the endothelium-independent vasodilator, sodium nitroprusside (B). ECMR^−/− (C) and amiloride (D) improved flow-induced mesenteric artery dilation.n=4 to 5 per group. *P<0.05 compared with WD ECMR^+/+ or ECMR^+/+.

Figure 4. ECMR^−/− prevents WD-impaired eNOS activation and endothelial dependent aortic relaxation

Vasodilator responses of isolated aortic rings to the endothelium-dependent dilators, acetylcholine (A) and insulin (B). (C) The expression and activation of Akt and eNOS were performed with immunoblotting. (B) Quantitative analysis of protein expression in p-Akt and p-eNOS. n=4 to 5 per group. *P<0.05 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.

Figure 5. WD-induced aortic oxidative stress and maladaptive pro-inflammatory cytokines are ameliorated in ECMR^−/− female mice

(A) Western blot for 3-NT, a marker of oxidant stress from accumulation of oxidant peroxynitrite (ONOO⁻). (B) mRNA expression of Nox2 and p22phox in aortic tissues. (C) Expression of M1/M2 macrophage marker CD86, CD11c, CD206, and IL10. (D) WD ECMR^−/− increased M2 macrophage marker IL10 and CD206 mRNA expression in WD fed mice as measured by real-time PCR. n=4 per group. *P<0.01 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.

Figure 6. WD-induced aortic remodeling and ultrastructural abnormalities are prevented in in ECMR^−/− female mice

Representative micrographs show medial wall thickening staining with Verhoeff-Van Gieson (A) and periaortic fibrosis staining with picrosirius red staining (B). (C) ECMR^−/− prevents WD-induced increases in thickened electron dense plasmalemma and free ribosomes, which contributed to EC stiffness. MTO= microtuble organizing center; IEL = internal elastic lamina; VVO=vesiculovacuolar organelles. Magnification X 10,000; bar = 0.5 μm. (D) ECMR^−/− prevents WD-induced upregulation of p-Erk 1/2. n=4-6 per group. *P<0.05 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.