Microvascular angiotensin II type 2 receptor function is enhanced in young females and declines in a model of murine aging

Abstract

Introduction: Angiotensin II (AngII) affects cardiovascular health, mediating impacts through AngII type 1 (AT1R) and type 2 (AT2R) receptors. The present study investigated sex and aging-related differences in microvascular AngII receptor function in mice and humans.

Methods: Mesenteric resistance arteries (MRA) were isolated from 3-, 12-, and 18-month-old female and male C57/Bl6 mice. Wire myography was used to measure vasoconstriction to AngII and vasodilation to an AT2R agonist (compound 21, C21). Seven healthy adults (3 premenopausal women and 4 age-matched men) were recruited to participate in a study measuring cutaneous microvascular vasoconstriction to AngII in the presence and absence of 10 μM PD123319, an AT2R antagonist.

Results: In murine MRA, AngII-induced constriction increases by 18 months in females and by 12 months in males. AT2R-mediated vasodilation was reduced with age in females only, which corresponds with a female-specific decrease in mesenteric AT2R mRNA expression. AT2R inhibition enhances AngII-induced constriction in young female, but not male, mice. Clinical data support that premenopausal women have attenuated AngII constriction vs. men, which is abrogated by AT2R inhibition. AT2R expression is greater in primary aortic smooth muscle cells, but not endothelial cells, from young women compared with men.

Conclusions: These data demonstrate enhanced microvascular AT2R function in young female mice and young women. There is a female-specific loss of AT2R function with age in mice, concomitant with declining AT2R expression. These findings implicate AT2R as a sex-specific target for microvascular dysfunction and aging-associated cardiovascular disease.

Keywords: Microvascular function; aging; angiotensin II type 2 receptor; cutaneous; sex differences; skin blood flow.

Figure 1.

Angiotensin II-induced vasoconstriction increases later in life in female compared with male mice. Second- and third-order mesenteric resistance arteries were isolated from 3-, 12-, and 18-month-old C57/Bl6 mice. Four separate segments from each mouse were hung in a wire myograph and administered a different concentration of angiotensin II (1 × 10⁻⁹ to 1 × 10⁻⁷ M). Microvascular constriction was measured in vessels from (A) females and (B) males in response to angiotensin II concentrations by wire myography. Data are expressed as raw force in milliNewtons (mN) from baseline. N = 3–4/group (all female groups and 3-month-old males = 3/group; 12- and 18-month-old males = 4/group). The main effects of concentration and age and the interaction effect of these two factors were assessed via repeated measures two-way ANOVA with Tukey post hoc testing where appropriate. Data are means ± SEM, *P < 0.05, 18-month-old females vs. all other female groups. **P < 0.01, 18-month-old females vs. all other female groups. ^#P < 0.05, 3-month-old males vs. 12-month-old males.

Figure 2.

AT2R-mediated vasodilation declines with age in female mice only. Second- and third-order mesenteric resistance arteries were isolated from 3-, 12-, and 18-month-old C57/Bl6 (A) female and (B) male mice. Vessels were pre-treated with losartan for 30 min to block angiotensin II type 1 receptors. Following preconstriction to phenylephrine (PE), vasorelaxation in response to Compound 21 (AT2R agonist, 1 × 10⁻¹² to 1 × 10⁻⁷ M) was determined by wire myography. Data are expressed as percent relaxation from PE preconsriction. N = 3–7/group (18-month-old males and females = 3/group; 3-month-old females and males and 12-month-old males = 4/group; 12-month-old females = 7/group). The main effects of concentration and age and the interaction effect of these two factors were assessed via repeated measures two-way ANOVA with Tukey post hoc testing where appropriate. Data are means ± SEM, *P < 0.05, 3-month-old females vs. 18-month-old females; **P < 0.01, 3-month-old females vs. 18-month-old females; ^#P < 0.05, 12-month-old females vs. 18-month-old females.

Figure 3.

Mesenteric resistance artery AT2R mRNA declines with age in female mice only. Total RNA was extracted from mesenteric resistance arteries, and qRT/PCR was performed to quantify angiotensin II type 2 [AT2R, (A and B)] and type 1b [AT1BR, (C and D)] receptor mRNA expression. N = 5–6/group. Differences were assessed via one-way ANOVA with Tukey post hoc testing where appropriate. Data are means ± SEM, *P < 0.05; **P < 0.01.

Figure 4.

Angiotensin II-induced vasoconstriction is attenuated in young female compared with male mice, and this difference is abolished with AT2R inhibition. Second- and third-order mesenteric resistance arteries were isolated from 3-month-old C57/Bl6 female and male mice. Vessels were pre-treated with vehicle (circles) or 10 μM PD123319 (squares), an AT2R antagonist. Vessel constriction in response to AngII (1 × 10⁻⁷ M) was determined by wire myography. Data are expressed as raw force in milliNewtons (mN). N = 8–10/group. The main effects of treatment and sex and the interaction effect of these two factors were assessed via two-way ANOVA with Tukey post hoc testing where appropriate. Data are means ± SEM, *P < 0.05.

Figure 5.

Angiotensin II-induced vasoconstriction is attenuated in premenopausal women compared with men, and this difference is abolished with AT2R inhibition. Forearm cutaneous vascular conductance was assessed to measure microvascular constriction in response to increasing concentrations of AngII (1 × 10⁻⁹ to 1 × 10⁻⁴ M) in young, premenopausal women (N = 3, red circles, average age: 28 ± 2 years) and young, age-matched men (N = 4, blue squares, average age: 30 ± 3 years). Local skin sites were pre-treated with (A) lactated Ringer’s (vehicle control) or (B) an AT2R antagonist (1 μM PD123319). Cutaneous vascular conductance (red blood cell flux/mean arterial pressure) is expressed as a percentage of baseline. The decline in cutaneous vascular conductance in response to AngII corresponds to the degree of vasoconstriction of the cutaneous microvasculature. The main effects of concentration and sex and the interaction effect of these two factors were assessed via repeated measures two-way ANOVA with Tukey post hoc testing where appropriate. Data are means ± SEM, **P < 0.01.

Figure 6.

AT2R expression in human aortic smooth muscle cells is greater in premenopausal women vs. men. The expression of angiotensin II type 2 [AT2R, (A and B)] and type 1 [AT1R, (C and D)] was quantified in primary human aortic smooth muscle cells (A and C) and endothelial cells (B and D). Samples are from adult women (average age: 37 ± 1 years) and men (average age: 34 ± 2 years) donors. Representative immunoblots and quantification are shown. N = 3–8 samples per group. Differences were assessed via the student’s t-test. Data are means ± SEM for protein expression normalized to GAPDH, *P < 0.05.