Western-style diet modulates contractile responses to phenylephrine differently in mesenteric arteries from senescence-accelerated prone (SAMP8) and resistant (SAMR1) mice

Abstract

The influence of two known cardiovascular risk factors, aging and consumption of a high-fat diet, on vascular mesenteric artery reactivity was examined in a mouse model of accelerated senescence (SAM). Five-month-old SAM prone (SAMP8) and resistant (SAMR1) female mice were fed a Western-type high-fat diet (WD; 8 weeks). Mesenteric arteries were dissected, and vascular reactivity, protein and messenger RNA expression, superoxide anion (O 2 (·-) ) and hydrogen peroxide formation were evaluated by wire myography, immunofluorescence, RT-qPCR, ethidium fluorescence and ferric-xylenol orange, respectively. Contraction to KCl and relaxation to acetylcholine remained unchanged irrespective of senescence and diet. Although similar contractions to phenylephrine were observed in SAMR1 and SAMP8, accelerated senescence was associated with decreased eNOS and nNOS and increased O 2 (·-) synthesis. Senescence-related alterations were compensated, at least partly, by the contribution of NO derived from iNOS and the enhanced endogenous antioxidant capacity of superoxide dismutase 1 to maintain vasoconstriction. Administration of a WD induced qualitatively different alterations in phenylephrine contractions of mesenteric arteries from SAMR1 and SAMP8. SAMR1 showed increased contractions partly as a result of decreased NO availability generated by decreased eNOS and nNOS and enhanced O 2 (·-) formation. In contrast, WD feeding in SAMP8 resulted in reduced contractions due to, at least in part, the increased functional participation of iNOS-derived NO. In conclusion, senescence-dependent intrinsic alterations during early stages of vascular senescence may promote vascular adaptation and predispose to further changes in response to high-fat intake, which may lead to the progression of aging-related cardiovascular disease, whereas young subjects lack the capacity for this adaptation.

Fig. 1

Influence of senescence and diet on the concentration–response curve to phenylephrine (a) and acetylcholine (b) in mesenteric arteries from female SAMR1 and SAMP8 mice. WD Western diet. Results are the mean ± SEM from SAMR1 (control 16–20; WD 10–14) and SAMP8 (control 14–16; WD 7–10) mice. *P < 0.05, **P < 0.01 by two-way ANOVA with Bonferroni’s post-test

Fig. 2

Influence of senescence and diet on the concentration–response curve to phenylephrine in mesenteric arteries from female SAMR1 and SAMP8 mice. The role of eNOS and iNOS was assessed with L-NAME (100 μM) (a, b) or 1400 W (10 μM) (c, d). WD Western diet. Results are the mean ± SEM from SAMR1 (control 6–16; WD 4–10) and SAMP8 (control 5–14; WD 5–7) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA with Bonferroni’s post-test

Fig. 3

Influence of senescence and diet on NOS expression in mesenteric arteries from female SAMR1 and SAMP8 mice. a Representative photomicrographs and quantification of eNOS, iNOS and nNOS immunofluorescence of confocal microscopic artery sections. Immunofluorescent signal (red) and natural autofluorescence of elastin (green) are shown. ADV adventitial layer, END endothelial layer, MED media layer, IEL internal elastic lamina, WD Western diet. Image size 238 × 238 μm. SAMR1 (control 5–6; WD 4–5) and SAMP8 (control 5–7; WD 4–5) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA with Bonferroni’s post-test. b Comparative analysis of mRNA levels of eNOS, iNOS and nNOS in mesenteric arteries from female SAMR1 and SAMP8 mice. mRNA levels are expressed as 2^−ΔCt using 18 S as internal control. Results are the mean ± SEM from SAMR1 (control 6; WD 6) and SAMP8 (control 6; WD 6) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA with Bonferroni’s post-test

Fig. 4

Influence of senescence and diet on the concentration–response curve to phenylephrine in mesenteric arteries from female SAMR1 (a) and SAMP8 (b) mice. The role of superoxide anion was assessed with tempol (1 mM). WD Western diet. Results are the mean ± SEM from SAMR1 (control 4–16; WD 5–10) and SAMP8 (control 8–14; WD 3–7) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA with Bonferroni’s post-test

Fig. 5

Influence of senescence and diet on superoxide anion formation in mesenteric arteries from female SAMR1 and SAMP8 mice. a Representative photomicrographs and quantification of fluorescence of confocal microscopic artery sections labeled with the oxidative dye dihydroethidium, which produces a red fluorescence when oxidized to ethidium bromide by superoxide anion. Natural autofluorescence of elastin (green) is also shown. ADV adventitial layer, END endothelial layer, MED media layer, IEL internal elastic lamina, WD Western diet. Image size 238 × 238 μm. Results are the mean ± SEM from SAMR1 (control 5; WD 5) and SAMP8 (control 5; WD 5) mice. *P < 0.05 by two-way ANOVA with Bonferroni’s post-test. b Representative photomicrographs and quantification of nitrotyrosine immunofluorescence of confocal microscopic artery sections. Immunofluorescent signal (red) and natural autofluorescence of elastin (green) are shown. ADV adventitial layer, END endothelial layer, MED media layer, IEL internal elastic lamina, WD Western diet. Image size 238 × 238 μm. SAMR1 (control 5; WD 5) and SAMP8 (control 5; WD 5) mice. *P < 0.05, **P < 0.01 by two-way ANOVA with Bonferroni’s post-test. c Comparative analysis of mRNA levels of the NAD(P)H oxidase subunit Nox-1, p22^phox and p47^phox. mRNA levels are expressed as 2^−ΔCt using 18 S as internal control. Results are the mean ± SEM from SAMR1 (control 6; WD 6) and SAMP8 (control 6; WD 6) mice. *P < 0.05, **P < 0.01 by two-way ANOVA with Bonferroni’s post-test

Fig. 6

Influence of senescence and diet on endogenous antioxidant capacity in mesenteric arteries from female SAMR1 and SAMP8 mice. a Representative photomicrographs and quantification of staining of microscopic artery sections. This assay is based on the conversion of Fe⁺² to Fe⁺³ in the presence of hydroperoxide, which in turn complexes with xylenol orange dye to yield a purple product. WD Western diet. Image size 858 × 858 μm. Results are the mean ± SEM from SAMR1 (control 5; WD 7) and SAMP8 (control 8; WD 11) mice. **P < 0.01, ***P < 0.001 by two-way ANOVA with Bonferroni’s post-test. b Comparative analysis of mRNA levels of SOD1, SOD2 and SOD3. mRNA levels are expressed as 2^−ΔCt using 18 S as internal control. Results are the mean ± SEM from SAMR1 (control 6; WD 6) and SAMP8 (control 6; WD 6) mice. **P < 0.01 by two-way ANOVA with Bonferroni’s post-test