Angiotensin-converting enzyme 2 deficiency in whole body or bone marrow-derived cells increases atherosclerosis in low-density lipoprotein receptor-/- mice

Abstract

Objective: The renin-angiotensin system contributes to atherosclerotic lesion formation. Angiotensin-converting enzyme 2 (ACE2) catabolizes angiotensin II (Ang II) to angiotensin 1-7 (Ang-(1-7)) to limit effects of the renin-angiotensin system. The purpose of this study was to define the role of ACE2 in atherosclerosis.

Methods and results: Male Ace2(-/y) mice in an low-density lipoprotein receptor-deficient background were fed a high-fat diet for 3 months. ACE2 deficiency increased atherosclerotic area (Ace2(+/y), 17 ± 1; Ace2(-/y), 23 ± 2 mm(2), P < 0.002). This increase was blunted by losartan. To determine whether leukocytic ACE2 influenced atherosclerosis, irradiated low-density lipoprotein receptor-deficient male mice were repopulated with bone marrow-derived cells from Ace2(+/y) or Ace2(-/y) mice and fed a high-fat diet for 3 months. ACE2 deficiency in bone marrow-derived cells increased atherosclerotic area (Ace2(+/y), 1.6 ± 0.3; Ace2(-/y), 2.8 ± 0.3 mm(2); P < 0.05). Macrophages from Ace2(-/y) mice exhibited increased Ang II secretion and elevated expression of inflammatory cytokines. Conditioned media from mouse peritoneal macrophages of Ace2(-/y) mice increased monocyte adhesion to human umbilical vein endothelial cells. Incubation of human umbilical vein endothelial cells with Ang II promoted monocyte adhesion, which was blocked by Ang-(1-7). Coinfusion of Ang-(1-7) with Ang II reduced atherosclerosis.

Conclusions: These results demonstrate that ACE2 deficiency in bone marrow-derived cells promotes atherosclerosis through regulation of Ang II/Ang-(1-7) peptides.

Figure 1

Whole body ACE2 deficiency increased atherosclerosis. A, ACE2 mRNA expression in kidneys from Ace2^+/+, Ace2^+/−, and Ace2^−/− Ldlr^−/− female mice (N = 6–8/genotype). B, En face lesion areas (%) in aortic arches (N = 10–13 mice/genotype). C, En face lesion areas (%) in aortic arches of Ace2^−/y male mice infused with saline or losartan during month 3 of HF feeding (N = 8 mice/group). D, Representative aortas from mice in each group stained with Oil red O. E, Lesion areas of Oil Red O stained sections from aortic sinuses (N = 6 mice/genotype). F, Quantification of CD68 immunostaining of sections from aortic sinuses (N = 4 mice/genotype). Circles represent individual mice with triangles representing group mean ± SEM. *, P<0.05 compared to Ace2^+/+ or ^+/y or compared to saline-infused Ace2^−/y mice.

Figure 2

ACE2 localized to lesional macrophages in Ldlr^−/− male mice. CD68 and ACE2 immunostaining in aortic sinus sections from Ldlr^−/− male mice. IgG staining (anti-rat, control for CD68) is illustrated on left. Boxed area is shown at higher magnification in lower panel. Scale bar represents 200 µm.

Figure 3

ACE2 deficiency in bone marrow-derived cells increased atherosclerosis in Ldlr^−/− male mice. A, En face lesion area (%) of aortic arches (N = 8–10 mice/genotype). B, Lesion areas of oil red O stained sections from aortic sinuses (N = 7–9 mice/genotype). C, Quantification of CD68 immunostaining in sections from aortic sinuses (N = 5 mice/genotype). Circles represent individual mice with triangles representing group mean ± SEM. D, Representative images of CD68 immunostaining in aortic sinus sections from Ace2^+/y or ^−/y mice. *, P<0.05 compared to Ace2+/y.

Figure 4

ACE2 deficiency promoted AngII release, chemokine receptor expression and inflammatory cytokine release from cultured peritoneal macrophages and increased monocyte adhesion to endothelial cells. A, AngII release from MPMs of Ace2^+/y or ^−/y mice. B, CCR2 mRNA abundance in MPMs from Ace2^+/y or ^−/y mice (N = 6 mice/genotype). C, IL-6 and PAI-1 protein levels released from MPMs of Ace2^+/y or ^−/y mice (N = 6 mice/genotype). D, Co-culture of MPMs from Ace2^−/y mice with HUVECs increased THP-1 monocyte adhesion compared to control (expressed as % THP-1 adhesion in macrophage media from Ace2^+/y mice). Co-incubation of MPMs from each genotype with losartan (1 µM; N = 3–6 mice/genotype) reduced monocyte adhesion, while co-incubation with D-Ala (5 µM; N = 3–6 mice/genotype) had no effect. *, P<0.05 compared to Ace2^+/y (A–D). †, P<0.05 compared to vehicle within genotype.

Figure 5

Ang-(1–7) functionally antagonized AngII-induced monocyte adhesion to HUVECs and AngII-induced atherosclerosis in Ldlr^−/− mice. A, Effect of AngII (1 µM), losartan (1 µM), Ang-(1–7) (1 µM), D-Ala + Ang-(1–7) (5 µM), alone or in combinations, on monocyte adhesion to HUVECs (N = 6/incubation). B, En face lesion area (%) of aortic arches from Ldlr−/− mice infused with AngII in the absence (AngII) or presence of Ang-(1–7) (N = 17 mice/treatment). C, CD68 quantification of aortic sinuses from stained sections (N = 5 mice/treatment). D, Lesion areas of Oil Red O stained sections from aortic sinuses (N = 8–9 mice/treatment). E, Representative aortas from mice in each group stained with Oil Red O. Data are mean ± SEM; *, P<0.05 compared to control cells (A) or AngII group (B,D, and E). Δ, P<0.05 compared to cells incubated with AngII.