Results from this study demonstrate that whole body deficiency of ACE2 increased the development of atherosclerosis in fat-fed Ldlr−/− mice. ACE2 localized to macrophage-rich regions of atherosclerotic lesions, and ACE2 enzymatic activity was evident in mouse peritoneal macrophages. Interestingly, deficiency of ACE2 in bone marrow-derived cells also promoted the development of atherosclerosis. While plasma concentrations of AngII were not markedly elevated in whole body ACE2 deficient mice, macrophages from ACE2 deficient mice released greater concentrations of AngII. Moreover, macrophages from ACE2 deficient mice exhibited increased expression and release of inflammatory cytokines, and promoted monocyte adhesion to endothelial cells. While Ang-(1–7) had no effect on monocyte adhesion by itself, this angiotensin peptide functionally antagonized AngII-induced stimulation of monocyte adhesion and lesion formation. These results demonstrate that ACE2 deficiency promotes atherosclerosis, and suggest that endogenous ACE2 protects against atherosclerosis by controlling the relative balance between AngII and Ang-(1–7) in pivotal cell types.
We found that cultured macrophages from ACE2 deficient mice release greater concentrations of AngII. In contrast, whole body deficiency of ACE2 had no effect on systemic concentrations of renin or AngII. Our results are in agreement with several studies that document no change in plasma or kidney angiotensin peptide concentrations in ACE2 deficient mice.21,22
However, it should be noted that a previous study reported that ACE2 deficiency increased systemic concentrations of AngII in mice experiencing heart failure.13
An interesting aspect of the present study is that whole body and bone marrow cell deficiency of ACE2 promoted atherosclerosis, but had no effect on the systemic RAS. These results suggest that ACE2 primarily influences concentrations of AngII in pivotal cell types involved in developing lesions.
Previous investigators demonstrated that whole body ACE2 deficiency had strain-dependent effects on blood pressure.19
In C57BL/6 mice, ACE2 deficiency resulted in a modest increase in blood pressure (~7 mm Hg).19
We did not observe an effect of ACE2 deficiency on systolic blood pressures in Ldlr−/−
mice. It is possible that ACE2 deficiency on an Ldlr−/−
background blunted blood pressure increases in C57BL/6 mice. Previous studies demonstrated that although AT1a receptor deficiency had divergent effects on systolic blood pressure in Ldlr−/−
mice, loss of AT1a receptor signaling resulted in pronounced reductions in atherosclerosis.3,7
Therefore, it is unlikely that blood pressure effects from ACE2 deficiency contributed to augmented atherosclerosis observed in the present study.
Our results are in agreement with previous studies demonstrating that ACE2 localized to macrophage-rich areas of atherosclerotic lesions from hypercholesterolemic rabbits.15
However, these results extend previous findings by demonstrating that murine macrophages exhibit ACE2 enzymatic activity and that macrophages from ACE2 deficient mice release greater concentrations of AngII. Similar to previous findings demonstrating reductions in thoracic aortic lesion areas following bone marrow transplantation in high fat fed Ldlr−/−
, in this study bone marrow transplantation reduced lesion areas in aortic arches of high fat-fed Ldlr−/−
mice of both genotypes. Even though background levels of atherosclerosis were reduced in bone marrow transplanted mice, chimeric Ldlr−/−
mice with ACE2 deficiency in bone marrow-derived cells exhibited increased atherosclerosis of a similar magnitude (~2-fold) as seen from whole body ACE2 deficiency. Moreover, chimeric mice with ACE2 deficiency in bone marrow-derived cells exhibited increased atherosclerosis in the absence of changes in the systemic RAS. These results suggest that local effects of ACE2 on bone marrow-derived cells, potentially macrophages, mediate effects of ACE2 deficiency to promote atherosclerosis.
Previous investigators demonstrated that adenoviral overexpression of murine ACE2 in rabbits subjected to endothelial injury and fed an atherogenic diet attenuated lesion progression.16
Similarly, recent studies demonstrated that adenoviral over-expression of ACE2 in atherosclerotic lesions of rabbits attenuated fatty streak formation.20
mice, adenoviral overexpression of ACE2 reduced atherosclerosis.17
Moreover, recent studies demonstrated that whole body deficiency of ACE2 in ApoE−/−
mice increased atherosclerosis.18
In agreement, results from this study demonstrate that whole body deficiency of ACE2 in Ldlr−/−
mice increased atherosclerosis. Collectively, these results support a pivotal role for ACE2 in developing atherosclerotic lesions.
Our studies extend previous findings by contrasting effects of whole body versus bone marrow deficiency of ACE2 on atherosclerosis. Results demonstrate a similar magnitude increase in lesion formation in whole body ACE2 deficient mice compared to chimeric mice lacking ACE2 in bone marrow derived cells. While these findings do not indicate a specific cell type responsible for augmented lesion formation in mice lacking ACE2 in bone marrow derived cells, macrophage rich regions of lesions from Ldlr−/−
mice stained positively for ACE2. In addition, MPMs exhibited ACE2 enzymatic activity, and macrophages from ACE2 deficient mice released greater concentrations of AngII. Recent studies demonstrated that aortas from ACE2 deficient ApoE−/−
mice exhibited increased cell adhesion when perfused with whole blood.18
Moreover, bone marrow derived macrophages from ACE2 deficient mice exhibited increased expression of inflammatory cytokines in response to LPS.18
Results from the present study demonstrate increased monocyte adhesion when HUVECs were co-cultured with MPMs from ACE2 deficient mice, and extend previous findings by demonstrating that these effects are AngII/AT1 receptor mediated.2
Moreover, in this study, in the absence of external stimuli (e.g., LPS)18
, ACE2 deficient MPMs exhibited increased expression and release of inflammatory cytokines. These results demonstrate that deficiency of ACE2 in pivotal cell types promotes inflammation and monocyte adhesion, two mechanisms invoked in lesion formation.
In the setting of ACE2 deficiency where AngII is formed but not catabolized to Ang-(1–7), our results using losartan support a prominent role for AngII effects at AT1 receptors to augment atherosclerosis. In contrast, in wild type mice both AngII and Ang-(1–7) are present to regulate lesion formation. Recent studies demonstrated that long-term infusion of Ang-(1–7) to ApoE−/−
mice reduced atherosclerosis.24
Moreover, recent studies demonstrated functional antagonism of AngII-mediated regulation of smooth muscle cell proliferation and migration by Ang-(1–7), supporting functional interactions between these angiotensin peptides that are regulated by ACE2.20
Similar to findings in smooth muscle cells, in the present study while Ang-(1–7) had no effect on monocyte adhesion when incubated alone with HUVECs, this peptide functionally antagonized effects of AngII to promote monocyte adhesion. Since effects of Ang-(1–7) to blunt AngII-induced monocyte adhesion were abolished by D-Ala, these findings implicate the mas receptor as the mediator of Ang-(1–7)-induced antagonism of AngII. Infusion of Ang-(1–7) with AngII markedly lowered atherosclerosis in Ldlr−/−
mice, supporting an in vivo interplay between these ACE2-regulated angiotensin peptides in the development of atherosclerosis.
In conclusion, the present study demonstrated that whole body deficiency of endogenous ACE2, as well as deficiency in bone marrow-derived cells, increased atherosclerosis in Ldlr−/− mice. Elevated macrophage concentrations of AngII in ACE2 deficient mice promoted expression of inflammatory cytokines and increased monocyte adhesion to endothelial cells. When both AngII and Ang-(1–7) were present, Ang-(1–7) functionally antagonized effects of AngII to promote monocyte adhesion and lesion development. These results demonstrate that regulation of local concentrations of AngII versus Ang-(1–7) in pivotal cell types by enzymes such as ACE2 influence developing atherosclerotic lesions.