Angiotensin converting enzyme 2/Ang-(1-7)/mas axis protects brain from ischemic injury with a tendency of age-dependence

Abstract

Background: The angiotensin (Ang) converting enzyme 2 (ACE2)/Ang-(1-7)/Mas receptor pathway is an important component of the renin-angiotensin system and has been suggested to exert beneficial effects in ischemic stroke.

Aims: This study explored whether the ACE2/Ang-(1-7)/Mas pathway has a protective effect on cerebral ischemic injury and whether this effect is affected by age.

Methods: We used three-month and eight-month transgenic mice with neural over-expression of ACE2 (SA) and their age-matched nontransgenic (NT) controls. Neurological deficits and ischemic stroke volume were determined following middle cerebral artery occlusion (MCAO). In oxygen and glucose deprivation (OGD) experiments on brain slices, the effects of the Mas receptor agonist (Ang1-7) or antagonist (A779) on tissue swelling, Nox2/Nox4 expression reactive oxygen species (ROS) production and cell death were measured.

Results: (1) Middle cerebral artery occlusion -induced ischemic injury and neurological deficit were reduced in SA mice, especially in eight-month animals; (2) OGD-induced tissue swelling and cell death were decreased in SA mice with a greater reduction seen in eight-month mice; (3) Ang-(1-7) and A779 had opposite effects on OGD-induced responses, which correlated with changes in Nox2/Nox4 expression and ROS production.

Conclusions: Angiotensin converting enzyme 2/Ang-(1-7)/Mas axis protects brain from ischemic injury via the Nox/ROS signaling pathway, with a greater effect in older animals.

Keywords: Aging; Angiotensin; Angiotensin-converting enzyme 2; Ischemic stroke; NADPH oxidase.

Figure 1

Neuronal over‐expression of ACE2 reduces MCAO‐induced cerebral injury in mice with enhanced efficacy in eight‐month mice. (A): representative TTC staining images of brain sections obtained 48 h after MCAO from NT and SA mice. (B): summarized data of infarct volumes. (C): summarized data of neurological deficit scores. SA: mice with neuronal over‐expression of ACE2; NT: wild‐type control mice; MCAO: middle cerebral artery occlusion; TTC: 2, 3, 5‐triphenyltetrazolium chloride; 3M: three‐month mice; 8M: eight‐month mice. *P < 0.05, NT vs. SA, ⁺ P < 0.05, vs. 3M, n = 7/group.

Figure 2

Neural over‐expression of ACE2 or activation of Ang1‐7/Mas receptor pathway protects brain from OGD‐induced tissue swelling with enhanced efficacy in eight‐month mice. (A): Representative figures demonstrate the IOS in alive brain tissue at basal and after OGD. OGD induced tissue swelling was indexed by the increase of IOS in CA1 and cortex. Increase of IOS intensity is indicated by color from black to white. (B): Summarized data of neural over‐expression of ACE2 effect on OGD‐induced brain tissue swelling. Data are mean ± SEM, n = 7/group, *P < 0.05, vs. NT, ⁺ P < 0.05, vs. 3M. (C): Summarized data of Ang1–7 (10 μm) effect on OGD‐induced brain tissue swelling. (D): Summarized data of A779 (10 μm) effect on OGD‐induced brain tissue swelling. IOS: intrinsic optical signal; OGD: oxygen and glucose deprivation; 3M: three‐month mice; 8M: eight‐month mice. Data are mean ± SEM, n = 7/group, *P < 0.05, vs. Vehicle, ⁺ P < 0.05, vs. 3M.

Figure 3

Neural over‐expression of ACE2 or activation of Ang1‐7/Mas receptor pathway protects brain from OGD‐induced cell death with enhanced efficacy in eight‐month mice. (A): Representative figures demonstrate the PI staining in control and OGD brain tissues. OGD‐induced cell death was indexed by the PI ‐positive cells in CA1 and cortex. The scale bars indicate 75 μm. (B): Summarized data of neural over‐expression of ACE2 effect on OGD‐induced brain cell death. Data are mean ±SEM, n = 7/group, *P < 0.05 vs. NT, ⁺ P < 0.05 vs. 3M. (C): Summarized data of Ang1–7 (10 μm) effect on OGD‐induced cell death. (D): Summarized data of A779 (10 μm) effect on OGD‐induced cell death. PI: propidium iodide; 3M: three‐month mice; 8M: eight‐month mice, n = 7/group, *P < 0.05 vs. Vehicle, ⁺ P < 0.05 vs. 3M.

Figure 4

Neural over‐expression of ACE2 decreases OGD‐induced Nox2 and Nox4 up‐regulation with enhanced efficacy in eight‐month mice. (A): Representative figures demonstrate the Nox2 and Nox4 expression in brain tissue in the control and after OGD. OGD‐induced Nox2 and Nox4 expression was indexed by Western Blots. (B, C): Summarized data. 3M: three‐month mice; 8M: eight‐month mice. Mean ± SEM, n = 6/group, *P < 0.05, vs. NT, ⁺ P < 0.05, vs. 3M, ^## P < 0.01, vs. Control.

Figure 5

Effect of Ang1‐7/Mas receptor pathway on OGD‐induced brain Nox2 and Nox4 expression. (A): Summarize date of Nox2 expression. (B): Summarize date of Nox4 expression. 3M: three‐month mice; 8M: eight‐month mice. Mean ± SEM, n = 6/group, *P < 0.05, vs. Vehicle, ⁺ P < 0.05, vs. 3M.

Figure 6

Neuronal over‐expression of ACE2 or activation of Ang1‐7/Mas receptor pathway reduces OGD‐induced ROS production in brain slices with enhanced efficacy in eight‐month mice. (A): Representative figures of DHE staining in the brain slices of 3M and 8M NT and SA mice. The scale bars indicate 100 μm. (B): Summarized data of neural over‐expression of ACE2 effect on OGD‐induced ROS production. n = 7/group, *P < 0.05 vs. NT, ⁺ P < 0.05 vs. 3M. (C): Summarized data of Ang1‐7 (10 μm) effect on OGD‐induced ROS production. (D): Summarized data of A779 (10 μm) effect on OGD‐induced ROS production. n = 7/group, *P < 0.05 vs. Vehicle, ⁺ P < 0.05 vs. 3M. ROS: reactive oxygen species; 3M: three‐month mice; 8M: eight‐month mice.