Mas receptor deficiency augments angiotensin II-induced atherosclerosis and aortic aneurysm ruptures in hypercholesterolemic male mice

Abstract

Objective: Previous studies demonstrated that deficiency of angiotensin-converting enzyme 2 (ACE2) augmented angiotensin II (AngII)-induced atherosclerosis and abdominal aortic aneurysm (AAA) formation in hypercholesterolemic mice. Effects of ACE2 deficiency could arise from increased concentrations of its substrate, AngII, or decreased concentrations of its product, angiotensin-(1-7) [Ang-(1-7)]. Infusion of Ang-(1-7), a Mas receptor (MasR) ligand, to hypercholesterolemic male mice reduced AngII-induced atherosclerosis, suggesting a protective role of the Ang-(1-7)/MasR axis. However, it is unclear whether endogenous Ang-(1-7) acts at MasR to influence AngII-induced vascular diseases. The purpose of this study was to define the role of MasR deficiency in AngII-induced atherosclerosis and AAA formation and severity in hypercholesterolemic male mice.

Methods: MasR^+/+ and MasR^-/- male mice on a low-density lipoprotein receptor-deficient (Ldlr^-/-) or apolipoprotein E-deficient (Apoe^-/-) background were infused with AngII at either 600 or 1000 ng/kg/min by osmotic minipump for 28 days. Atherosclerosis was quantified at study end point as percentage lesion surface area of the aortic arch in Ldlr^-/- mice. Abdominal aortic internal diameters were quantified by ultrasound, and maximal external AAA diameters were quantified at study end point. Blood pressure was quantified by radiotelemetry and a tail cuff-based technique. Serum cholesterol concentrations and vascular tissue characterization were examined at study end point.

Results: MasR deficiency did not influence body weight, systolic blood pressure at baseline and during AngII infusion, or serum cholesterol concentrations in either Apoe^-/- or Ldlr^-/- mice. MasR deficiency increased AngII-induced atherosclerosis in aortic arches of Ldlr^-/- mice (P < .05), associated with increased oxidative stress and apoptosis in aortic root sections (P < .05). MasR deficiency also augmented internal and external AAA diameters and increased aortic ruptures of both Ldlr^-/- and Apoe^-/- mice (P < .05). These effects were associated with increased elastin breaks and T-lymphocyte and macrophage accumulation into abdominal aortas of AngII-infused MasR-deficient mice (P < .05).

Conclusions: These results demonstrate that MasR deficiency augmented AngII-induced atherosclerosis and AAA rupture through mechanisms involving increased oxidative stress, inflammation, and apoptosis, suggesting that MasR activation may provide therapeutic efficacy against vascular diseases.

Keywords: Aneurysm; Angiotensin-(1-7); Atherosclerosis; Mas receptor; Rupture.

Figure 1.

MasR deficiency increased AngII-induced atherosclerosis in Ldlr−/− and Apoe−/−male mice. En face analysis of aortic arches of MasR+/+ and MasR−/−/Ldlr−/− and Apoe−/− mice. Figure 1A, Representative images of the aortic arch from mice of each genotype (scale bar represents 5 mm). Figure 1B and 1C, Percent atherosclerosis lesion surface area in Ldlr−/− and Apoe−/− mice. Data are mean ± SEM from n=3-7 mice/group; *, P<0.05.

Figure 2.

Elevations in oxidative stress and apoptosis in aortic sinus tissue sections from MasR-deficient Ldlr−/− mice infused with AngII. Figure 2: Representative DHE staining (red, A) with quantification (B) from 2-3 aortic sinus tissue sections from n = 3 mice/genotype. Representative TUNEL staining (green, A) with quantification (C) of percent TUNEL positive cells in aortic sinus sections from mice of each genotype. Scale bar represents 50 microns. Data are mean ± SEM from n=3 sections/mouse, n=3 mice/genotype; *, P<0.05.

Figure 3.

MasR deficiency augmented formation and severity of AngII-induced AAAs. A,B: In vivo ultrasound abdominal aortic lumen diameters on day 27 of AngII infusion in Ldlr−/− (A; n=3-7 mice/group) and Apoe−/− (B, n=7-11 mice/group) mice. C,D: AAA incidence in AngII-infused Ldlr−/− (C) and Apoe−/− (D) mice. Numbers of mice/genotype are illustrated within columns for each group, while percent incidence is illustrated above each column. E,F: Maximal external AAA diameters of AngII-infused Ldlr−/− (E) and Apoe−/− (F) mice. G,H: Percent survival over days of AngII infusion in Ldlr−/− (G) and Apoe−/− (H) mice. Data are mean ± SEM. *, P<0.05.

Figure 4.

MasR deficiency increased elastin fragmentation and immune cell accumulation in AAA tissue sections. A, Representative Movat staining in AAA tissue sections from MasR+/+ or MasR−/− (KO) Apoe−/− mice infused with AngII. Nuclei and elastin stain black, smooth muscle cells stain red, blue staining indicates ground substance, and yellow staining indicates collagen. Boxes at lower magnification (100×) are magnified at 400× to illustrate fragmentation of elastin. B, Quantification of elastin fragmentation in AAA tissue sections from n=5-7 mice/genotype. C, Representative T-lymphocyte (CD3) immunostaining in AAA tissue sections from mice of each genotype. D, Quantification of CD3 positive T-lymphocyte staining per section. E, Representative macrophage (F4/80) immunostaining in AAA tissue sections from mice of each genotype. F, Quantification of F4/80 positive macrophage staining per section. Scale bar represents 100 microns. Data are mean ± SEM. *, P<0.05.

Figure 5.

MasR deficiency increases atherosclerosis and AAAs in hyperlipidemic male mice. Summary of findings in both atherosclerosis and AAAs with MasR-deficient mice. With MasR deficiency, the balance of angiotensin peptides favors a pro-inflammatory, pro-apoptotic, pro-atherogenic environment with increased AAA formation and rupture.