Neuronal over-expression of ACE2 protects brain from ischemia-induced damage

Abstract

Angiotensin (Ang) II exaggerates cerebral injury in ischemic damage. Angiotensin-converting enzyme type 2 (ACE2) converts Ang II into Ang (1-7) and thus, may protect against the effects of Ang II. We hypothesized that neuronal ACE2 over-expression decreases ischemic stroke in mice with Ang II overproduction. Human renin and angiotensinogen double transgenic (RA) mice and RA mice with neuronal over-expression of ACE2 (SARA) were used for the study. The mean arterial pressure (MAP) was calculated from telemetry-recorded blood pressure (BP). SARA mice were infused peripherally with Norepinephrine to "clamp" the BP, or intracerebroventricularly-infused with a Mas receptor antagonist (A-779). Middle cerebral artery occlusion (MCAO) surgery was performed to induce permanent focal ischemic stroke. Cerebral blood flow (CBF) and neurological function were determined. Two days after surgery, brain samples were collected for various analyses. Results showed: 1) When compared to chronically hypertensive RA mice, SARA mice had lower basal MAP, less MCAO-induced infarct volume, and increased CBF, neurological function and cerebral microvascular density in the peri-infarct area; 2) These changes in SARA mice were not altered after MAP "clamping", but partially reversed by brain infusion of A-779; 3) Ang (1-7)/Ang II ratio, angiogenic factors, endothelial nitric oxide synthase (eNOS) expression and nitric oxide production were increased, whereas, NADPH oxidase subunits and reactive oxygen species were decreased in the brain of SARA mice. ACE2 protects brain from ischemic injury via the regulation of NADPH oxidase/eNOS pathways by changing Ang (1-7)/Ang II ratio, independently of MAP changes.

Keywords: Angiotensin II; Angiotensin-converting enzyme type 2; Blood pressure; Ischemic stroke; Oxidative stress.

Fig. 1

Experimental procedure and time flow chart. DHE: dihydroethidium; ROS: reactive oxygen species; NO: nitric oxide; eNOS: endothelial nitric oxide synthase; Nox: NADPH oxidase; aCSF: artificial cerebrospinal fluid; NE: norepinephrine; CBF: cerebral blood flow; cMVD: cerebral microvascular density; icv: intracerebrolventricular; NDS: neurologic deficit scores.

Fig. 2

Brain angiogenic cytokines in RA and SARA mice. The level of angiogenic factors was determined by ELISA kit. The levels of angiogenic factors are increased in SARA mice. *P<0.05, **P<0.01 vs. RA, n=10/group.

Fig. 3

Levels of ROS and NO in RA and SARA mice. A, The representative pictures of ROS and NO production in the brain (cortex area) of RA and SARA mice. ROS and NO production were measured by DHE staining (red) and DAF-FM DA staining (green), respectively. Scale bars: 200 µm. B, Summarized data on ROS and NO production. *P<0.05, **P<0.01 vs. RA, n=10/group. ROS: reactive oxygen species; DHE: dihydroethidium; NO: nitric oxide; DAF-FM DA: 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate.

Fig. 4

Expressions of eNOS, Nox2, 4 in RA and SARA mice. Brains from RA and SARA mice were removed to isolate the cerebral blood vessel and vessel-depleted brain parenchyma. The expressions of eNOS, Nox2,4 were analyzed by Western blot. A, Representative bands for eNOS and Nox 2,4 expressions in the brain of RA and SARA mice. B, Summarized data on eNOS and Nox 2,4 expressions. *P<0.05, **P<0.01 vs. RA, n=10/group.

Fig. 5

MAP levels of RA and SARA mice with or without “clamping” before and after MCAO surgery. A, Representative raw BP traces at 9AM before and after stroke. B, Summarized data on MAP in different treatment groups during day and night time. *P<0.05 vs. RA vehicle; ⁺P<0.05 vs. SARA vehicle, n=10/group. MAP: mean arterial pressure; icv: intracerebroventricular.

Fig. 6

Effects of neuronal over-expression of ACE2 on ischemic cerebral injury and neurological function. A, Representative Fluoro-J staining images in the peri-infarct area of RA mice and SARA mice following MCAO. Scale bars: 3.5 mm. B, MCAO-induced cerebral injury in RA mice and SARA mice before or after BP “clamping”. BP “clamping” was applied to SARA mice. C, The NDS of SARA mice and RA mice following MCAO. *P<0.05, **P<0.01 vs. RA vehicle; ⁺P<0.05, ⁺⁺P<0.01 vs. vehicle, ^#P<0.05 vs. SARA “clamping”, n=10/group. NDS: neurological deficit scores.

Fig. 7

Effects of neuronal over-expression of ACE2 on CBF and cMVD after MCAO-induced ischemia. A, The relative CBF in the peri-infarct area of SARA mice and RA mice following MCAO. *P<0.05 vs. RA vehicle; ⁺P<0.05 vs. vehicle, ^#P<0.05 vs. SARA “clamping”. B, Representative pictures of CD31 staining (red) for cMVD in each group. Scale bars: 50 µm. C, Summarized data on cMVD. BP “clamping” was applied to SARA mice. **P<0.01 vs. contralateral; ⁺P<0.05 vs. RA mice, ^#P<0.05 vs. vehicle, n=10/group. CBF: cerebral blood flow; cMVD: cerebral microvascular density; icv: intracerebroventricular.