Impact of ACE2 deficiency and oxidative stress on cerebrovascular function with aging

Abstract

Background and purpose: Angiotensin II produces oxidative stress and endothelial dysfunction in cerebral arteries, and angiotensin II type I receptors may play a role in longevity and vascular aging. Angiotensin-converting enzyme type 2 (ACE2) converts angiotensin II to angiotensin (1-7) and thus, may protect against effects of angiotensin II. We hypothesized that ACE2 deficiency increases oxidative stress and endothelial dysfunction in cerebral arteries and examined the role of ACE2 in age-related cerebrovascular dysfunction.

Methods: Endothelial function, expression of angiotensin system components, NADPH oxidase subunits, and proinflammatory cytokines were examined in cerebral arteries from adult (12 months old) and old (24 months old) ACE2 knockout (KO) and wild-type (WT) mice. The superoxide scavenger tempol was used to examine the role of oxidative stress on endothelial function.

Results: Vasodilatation to acetylcholine was impaired in adult ACE2 KO (24±6% [mean±SE]) compared with WT mice (52±7%; P<0.05). In old mice, vasodilatation to acetylcholine was impaired in WT mice (29±6%) and severely impaired in ACE2 KO mice (7±5%). Tempol improved endothelial function in adult and old ACE2 KO and WT mice. Aging increased mRNA for tumor necrosis factor-α in WT mice, and significantly increased mRNA levels of NAPDH oxidase 2, p47(phox), and Regulator of calcineurin 1 in both ACE2 KO and WT mice. mRNA levels of angiotensin system components did not change during aging.

Conclusions: ACE2 deficiency impaired endothelial function in cerebral arteries from adult mice and augmented endothelial dysfunction during aging. Oxidative stress plays a critical role in cerebrovascular dysfunction induced by ACE2 deficiency and aging.

Figure 1

ACE2 expression in basilar arteries and kidneys. Panel A: ACE2 staining in basilar artery, especially in the smooth muscle layer of adult WT mice. ACE staining was also seen in the epithelium of renal tubules in adult and old WT mice. Staining was absent in sections from ACE2 KO mice. Similar findings were observed in 3 mice from each group. Panel B: Western blots showing ACE2 expression in kidney homogenates from WT mice, and absence of protein in the ACE2 KO mouse. ACE2 staining was not detected by western blotting in brain arteries.

Figure 2

Effects of ACE2 deficiency and oxidative stress on vascular function. Panel A: Vasodilatation to acetylcholine (ACh) in adult ACE2 WT(●, n=5) and KO (○, n=5) mice. Role of oxidative stress was examined after incubation with tempol of basilar arteries from adult ACE2 WT (■) and KO (□) mice. Panel B: Vasodilatation to acetylcholine (ACh), was examined in old ACE2 WT(▲, n=5) and KO (△, n=6) mice. Tempol was also added to arteries from old ACE2 WT (◆) and KO (◇) mice. Values are mean ±SE, *p<0.05.

Figure 3

Effect of aging on expression of NADPH oxidase subunits. Relative expression levels of Nox2 and p47phox mRNA in cerebral arteries from adult (black bars) and old (gray bars) wild type or ACE2 KO mice. Values are mean ±SE (n=7/group), *p<0.05 vs adult mice.

Figure 4

Plasma levels of angiotensin peptides. Panel A: Angiotensin II levels in plasma from adult WT (n=7), adult ACE2 KO (n=6), old WT (n=7) and old ACE2 KO (n=7) mice. Panel B: Angiotensin 1–7 levels in plasma from adult WT (n=6), adult ACE2 KO (n=6), old WT (n=6) and old ACE2 KO (n=7) mice. There were no significant differences in plasma peptide levels between groups. Values are mean ±SE.