Age-related changes in vascular responses to angiotensin-(1-7) in female mice

Figures

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  Figure 1.. Ang-(1-7) induces a dose-dependent vasodilatory effect in aorta of both young and elderly male mice. (a) Concentration–response curves to Ang-(1-7) (10−10 to 10−6M) in thoracic aortic rings from male mice. Ang-(1-7) induces a dose-dependent vasodilation in aortic rings from both young (n=9) and old (n=8) male mice. (b) Concentration–response curves to acetylcholine (10−9 to 10−4 M) in thoracic aortic rings from young (n=9) and old (n=8) males. (c) Concentration–response curves to sodium nitroprusside (10−10 to 10−6 M) in thoracic aortic rings from young (n=9) and old (n=8) males. The endothelial function of thoracic aorta was preserved in response to both vasodilatory drugs. ▲: young males; △: old males. ANOVA followed by Newman–Keuls Multiple Comparison post hoc test was performed as statistical analysis. Each point represents the mean ± SEM. *p < 0.05.

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  Figure 2.. Absence of Ang-(1-7) vasodilatory response in aortic rings from old females. (a) Concentration–response curves to Ang-(1-7) (10−10 to 10−6 M) in thoracic aortic rings from young (n=10), old (n=16) and old E2-treated (n=5) female mice. Ang-(1-7) induced a concentration-dependent vasodilator effect in young females, but this effect was absent in old females. E2 treatment for seven days (1µg/day) in old females restored Ang-(1-7) response. (b) Concentration–response curves to acetylcholine (10−9 to 10−4 M) in thoracic aortic rings from young (n=5), old (n=8) and old female E2-treated (n=5) mice. (c) Concentration–response curves to Ang-(1-7) (10−10 to 10−6 M) in thoracic aortic rings from young (n=6), young OVX (n=6) and young OVX+E2 (n=6) female mice. Similar to old females, ovariectomized animals have no vasodilatory responses to Ang-(1-7). This response was recovered after E2 replacement. •: young females; ■: young females OVX; □: young females OVX+E2; ○: old females; ◑ : old females E2-treated. ANOVA followed by Newman–Keuls Multiple Comparison post hoc test was performed as statistical analysis. Each point represents the mean ± SEM. *p < 005.

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  Figure 3.. Decreased NO production in aortic rings from ageing females. To evaluate the effect of E2 on NO production, aortic arch longitudinal sections from young, elderly and elderly+E2 were analyzed. (a) Representative microphotographs using DAF-FM (green) in aortic arch from young (n=4), old (n=4) and old E2-treated (seven days, 1 µg/day, n=3) female mice. (b) Graph represents NO production by endothelial cells from aortic rings. Elderly females presented a decreased NO production compared with young females. Statistical analyses: ANOVA followed by Newman–Keuls Multiple Comparison post hoc test was performed as statistical analysis. #p < 0.05 compared to the control group. DAF-FM: 4,5-diaminofluorescein-diacetate probe; L: lumen.

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  Figure 4.. Ageing-related changes in vascular oxidative stress balance in female aorta. (a–d) Representative Western blotting (top) and the average densitometry values (bottom). Western blotting analysis of abdominal aortic protein homogenates from young (n=7), old (n=7) and old E2-treated (seven days, 1 µg/day, n=5) female mice. (a–c) Elderly females have significantly increased Mas receptor, SOD2 and catalase expression in abdominal aorta. This high expression was rescued in E2-treated old females, except for catalase. (d) NRF-2 and NOX2/gp91 upregulation was also rescued by E2 treatment. ANOVA followed by Newman–Keuls Multiple Comparison post hoc test was performed as statistical analysis. *p < 0.05 compared with the other groups. #p < 0.05 compared with the control group.

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  Figure 5.. Estradiol treatment rescues increased superoxide concentration in aorta from elderly female mice. (a) Upper panel: representative microphotographs using DHE-derived fluorescence (red) of aortic rings from young (n=4), old (n=4) and old E2-treated (seven days, 1 µg/day, n=3) female mice. Nuclei were stained with DAPI (blue). Scale bar = 50 µm. (b) Elderly females have significantly increased DHE labelling in abdominal aorta. This high expression was rescued in E2-treated old females. Statistical analyses: ANOVA followed by Newman–Keuls Multiple Comparison post hoc test was performed as statistical analysis. *p < 0.05 compared with the other groups. DHE: dihydroethidium.

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  Figure 6.. Proposed model of E2 effects involved in the regulation of vascular responses in ageing. Elderly females have less Ang-(1-7)-induced vasoprotective effects related to low levels of E2. To develop a mechanism to prevent increase in pressure levels, there is an increase in Mas receptor expression and its pathways in old females, for example, increased expression of SOD2/NRF-2/NOX2, a pathway involved in the control of the reactive oxygen species formation, which would be deleterious to the vessel. Despite these adaptive changes, vascular responsiveness remained inadequate in these females and upregulation of the Mas receptor axis was not able to overcome the deleterious effects induced by E2 absence. By replacing E2, there is a reversal of vascular nonresponsiveness to Ang-(1-7) and reestablishment of changes in Mas receptor and SOD2/NRF-2/NOX2 expression present in aorta from these old females. Ang-(1-7): angiotensin-(1-7); E2: estradiol; SOD: superoxide dismutase 2; NRF-2: nuclear factor erythroid 2-related factor 2; NOX2/gp91: isoform 2 from membrane-bound enzyme complex (NADPH).