Epithelial sodium channels in endothelial cells mediate diet-induced endothelium stiffness and impaired vascular relaxation in obese female mice

Abstract

Objective: Mineralocorticoid receptor activation of the epithelial sodium channel in endothelial cells (ECs) (EnNaC) is accompanied by aldosterone induced endothelial stiffening and impaired nitric oxide (NO)-mediated arterial relaxation. Recent data support enhanced activity of the alpha subunit of EnNaC (αEnNaC) mediates this aldosterone induced endothelial stiffening and associated endothelial NO synthase (eNOS) activation. There is mounting evidence that diet induced obesity diminishes expression and activation of AMP-activated protein kinase α (AMPKα), sirtuin 1 (Sirt1), which would be expected to lead to impaired downstream eNOS activation. Thereby, we posited that enhanced EnNaC activation contributes to diet induced obesity related increases in stiffness of the endothelium and diminished NO mediated vascular relaxation by increasing oxidative stress and related inhibition of AMPKα, Sirt1, and associated eNOS inactivation.

Materials/methods: Sixteen to twenty week-old αEnNaC knockout (αEnNaC^-/-) and wild type littermate (EnNaC^+/+) female mice were fed a mouse chow or an obesogenic western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. Sodium currents of ECs, endothelial stiffness and NO mediated aortic relaxation were examined along with indices of aortic oxidative stress, vascular remodeling and fibrosis.

Results: Enhanced EnNaC activation-mediated WD-induced increases in sodium currents in isolated lung ECs, increased endothelial stiffness and impaired aortic endothelium-dependent relaxation to acetylcholine (10^-9-10^-4 mol/L). These abnormalities occurred in conjunction with WD-mediated aortic tissue oxidative stress, inflammation, and decreased activation of AMPKα, Sirt1, and downstream eNOS were substantially mitigated in αEnNaC^-/- mice. Importantly, αEnNaC^-/- prevented WD induced increases in endothelial stiffness and related impairment of endothelium-dependent relaxation as well as aortic fibrosis and remodeling. However, EnNaC signaling was not involved in diet-induced abnormal expression of adipokines and CYP11b2 in abdominal aortic perivascular adipose tissue.

Conclusion: These data suggest that endothelial specific EnNaC activation mediates WD-induced endothelial stiffness, impaired eNOS activation, aortic fibrosis and remodeling through increased aortic oxidative stress and increased inflammation related to a reduction of AMPKα and Sirt 1 mediated eNOS phosphorylation/activation and NO production.

Keywords: Endothelial sodium channels; Endothelial stiffness; Fuel sensing kinases; Nitric oxide; Obesity; Oxidative stress.

Fig 1.

αEnNaC^−/− mice prevents WD-induced increases of inward Na⁺ currents on isolated lung ECs. Na⁺ current tracings (A), group I-V curves of Na⁺ currents (B), and comparison of peak inward Na+ currents at −80 mV (C) in αEnNaC^+/+ and αEnNaC^−/− mice fed a WD, n=31–38 cells.† p<0.01 compared with CD αEnNaC^+/+ group; ‡ p<0.01 compared with CD or WD αEnNaC^+/+ group in multiple comparison analysis.

Fig 2.

αEnNaC^−/− mice prevents WD-induced endothelium stiffness and impaired aortic relaxation. (A) The ex vivo measurement of endothelium stiffness by using atomic force microscopy. n=4–5. (B) Aortic pulse wave velocity measured in vivo, n=4–7. (C-D) Vasodilator responses of isolated aortic rings to the endothelium-dependent dilators, acetylcholine (C) and to the endothelium-independent vasodilator, sodium nitroprusside (D). n=5–9. † p<0.05 compared with CD αEnNaC^+/+ group; ‡ p<0.05 compared with WD αEnNaC^+/+ group in multiple comparison analysis.

Fig 3.

αEnNaC^−/− mice prevents WD-induced aortic oxidative stress. (A) Representative images of aortic sections stained for 3-nitrotyrosine (3-NT), a marker of oxidant stress from accumulation of oxidant peroxynitrite (ONOO⁻) with corresponding quantitative analysis (B). Scale bar = 50 μm, n=5–6. mRNA levels of Nox2 (C) in aortic tissues as measured by real-time PCR. n=5–7. † p<0.05 compared with CD αEnNaC^+/+ group; ‡ p<0.05 compared with WD αEnNaC^+/+ group in multiple comparison analysis.

Fig 4.

αEnNaC^−/− mice prevents WD-induced an increase of inflammation and reduction of AMPKα and eNOS activity and Sirt1 expression. (A) mRNA levels of ICAM-1, VCAM-1, IL6, MCP-1. (B) The protein abundance of AMPKα, eNOS, Sirt1 and GAPDH in aortic tissues were performed with immunoblotting. Quantitative analysis of protein abundance in p-AMPKα (C), p-eNOS (D), and Sirt1 (E). † p<0.05 compared with CD αEnNaC^+/+ group; ‡ p<0.05 compared with WD αEnNaC^+/+ group in multiple comparison analysis.

Fig 5.

αEnNaC^−/− mice prevents WD-induced aortic remodeling. Representative micrographs show aortic fibrosis staining with picrosirius red staining (A) and collagen 1 (B) with corresponding measures of average gray scale intensities.Scale bar = 50 μm, n=5–6. † p<0.05 compared with CD αEnNaC^+/+ group; ‡ p<0.05 compared with WD αEnNaC^+/+ group in multiple comparison analysis.

Fig. 6.

Deletion of αEnNaC in mice does not prevent WD-induced increases in inflammatory factors and CYP11b2 in abdominal aortic perivascular adipose tissue. (A) mRNA levels of adipokines in leptin, adiponectin, CD11b, IL6 and CD206. (B) mRNA levels of CYP11b2, ACE and ET-1. N=5–7. † p<0.05 compared with CD αEnNaC^+/+ group in multiple comparison analysis.

Fig 7.

Schematic diagram showing proposed mechanisms underlying the contribution of EnNaC to diet-induced increases in endothelial stiffness and impaired aortic NO mediated vascular relaxation.