Contributions of leukocyte angiotensin-converting enzyme to development of atherosclerosis

Abstract

Objective: This study determined the role of angiotensin-converting enzyme (ACE) on the development of angiotensin I-induced atherosclerosis and the contribution of leukocyte-specific expression of this enzyme.

Approach and results: To define the contribution of ACE-dependent activity to angiotensin II synthesis in atherosclerotic development, male low-density lipoprotein receptor(-/-) mice were fed a fat-enriched diet and infused with either angiotensin I or angiotensin II. The same infusion rate of these peptides had equivalent effects on atherosclerotic development. Coinfusion of an ACE inhibitor, enalapril, ablated angiotensin I-augmented atherosclerosis but had no effect on angiotensin II-induced lesion development. ACE protein was detected in several cell types in atherosclerotic lesions, with a predominance in macrophages. This cell type secreted angiotensin II, which was ablated by ACE inhibition. To study whether leukocyte ACE contributed to atherosclerosis, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with bone marrow-derived cells from either ACE(+/+) or ACE(-/-) mice and fed the fat-enriched diet for 12 weeks. Chimeric mice with ACE deficiency in bone marrow-derived cells had modestly reduced atherosclerotic lesions in aortic arches but had no effects in aortic roots.

Conclusions: ACE mediates angiotensin I-induced atherosclerosis, and ACE expression in leukocytes modestly contributes to atherosclerotic development in hypercholesterolemic mice.

Keywords: angiotensin-converting enzyme inhibitors; angiotensins; atherosclerosis; hypercholesterolemia; macrophages; renin.

Figure 1. Inhibition of ACE reduced AngI-induced atherosclerotic lesions

Percent atherosclerotic lesion area was measured on the intimal surface of aortic arches in LDL receptor -/- mice using an en face method. (A) Infusion of saline (n=19), AngI (n=18), or both AngI and enalapril (n=10). (B) Infusion of AngI (n=9), AngII (n=10), or both AngII and enalapril (n=8). Triangles represent values of individual mice, circles represent means, and bars are SEM. * denotes P<0.001 versus AngI infusion by one way ANOVA.

Figure 2. ACE protein was present in atherosclerotic lesions and was associated with multiple cell types

Immunostaining of (A) ACE (Goat anti-human ACE, 10 μg/ml), (B) CD68 (rat anti-mouse CD68, 5 μg/ml), (C) Smooth muscle α-actin (rabbit anti-α smooth muscle actin, 2.5 μg/ml), and (D) CD31 (biotin-conjugated rat anti-mouse CD31, 1 μg/ml) were performed on serial sections from aortic roots of male LDL receptor -/- mice fed a saturated fat-enriched diet for 12 weeks.

Figure 3. Macrophages secreted AngII and MCP-1 through an ACE-dependent mechanism

(A) AngII secreted from cultured wild-type peritoneal macrophages was separated by a high-performance liquid chromatography and measured by radioimmunoassay. (B) MCP-1 mRNA abundance in cultured peritoneal macrophages from ACE +/+ or -/- mice was determined using quantitative PCR. (C) MCP-1 protein released from cultured peritoneal macrophages of ACE +/+ or -/- mice were measured using an ELISA kit. * P<0.05.

Figure 4. ACE deficiency in bone marrow-derived cells reduced atherosclerosis in aortic arches, but not in aortic roots of LDL receptor -/- mice

(A) The presence of wild type or disrupted ACE allele in bone marrow-derived cells of chimeric mice was confirmed by PCR. (B) Percent lesion area in aortic arches was measured using an en face method in chimeric mice repopulated with bone marrow-derived cells from ACE +/+ (n=15) and ACE -/- (n=13) mice. (C) Lesion area in aortic roots was measured on serial cross-sections in mice repopulated with bone marrow-derived cells from ACE +/+ (n=11) and ACE -/- (n=10) mice. Triangles represent values of individual mice, circles represent means, and bars are SEM. * P<0.05 by Student's t-test.