Angiotensin-(1-7) induces cerebral ischaemic tolerance by promoting brain angiogenesis in a Mas/eNOS-dependent pathway

Abstract

Background and purpose: As a newer component of the renin-angiotensin system, angiotensin-(1-7) [Ang-(1-7) ] has been shown to facilitate angiogenesis and protect against ischaemic damage in peripheral tissues. However, the role of Ang-(1-7) in brain angiogenesis remains unclear. The aim of this study was to investigate whether Ang-(1-7) could promote angiogenesis in brain, thus inducing tolerance against focal cerebral ischaemia.

Experimental approach: Male Sprague-Dawley rats were i.c.v. infused with Ang-(1-7), A-779 (a Mas receptor antagonist), L-NIO, a specific endothelial NOS (eNOS) inhibitor, endostatin (an anti-angiogenic compound) or vehicle, alone or simultaneously, for 1-4 weeks. Capillary density, endothelial cell proliferation and key components of eNOS pathway in the brain were evaluated. Afterwards, rats were subjected to permanent middle cerebral artery occlusion (pMCAO), and regional cerebral blood flow (rCBF), infarct volume and neurological deficits were measured 24 h later.

Key results: Infusion of Ang-(1-7) for 4 weeks significantly increased brain capillary density via promoting endothelial cell proliferation, which was accompanied by eNOS activation and up-regulation of NO and VEGF in brain. These effects were abolished by A-779 or L-NIO. More importantly, Ang-(1-7) improved rCBF and decreased infarct volume and neurological deficits after pMCAO, which could be reversed by A-779, L-NIO or endostatin.

Conclusions and implications: This is the first evidence that Ang-(1-7) promotes brain angiogenesis via a Mas/eNOS-dependent pathway, which enhances tolerance against subsequent cerebral ischaemia. These findings highlight brain Ang-(1-7)/Mas signalling as a potential target in stroke prevention.

Figure 1

Ang-(1–7) increased cerebral capillary density and promoted endothelial cell proliferation in the ipsilateral hemisphere. Rats were infused with Ang-(1–7) for 1, 2 or 4 weeks, and the brain was removed and fixed. As indicated by the atlas on the top of this figure, three regions of interest (ROIs; size 500 × 500 μm) in the cerebral cortex (indicated by red box) were chosen from three separate brain coronal sections (−0.8, −1.8 and −2.8 mm from bregma). Capillary density in these ROIs of the ipsilateral hemisphere (A) and the contralateral hemisphere (B) was assessed by CD31 immunofluorescence staining. Bars: 100 μm. In addition, rats were infused with vehicle or Ang-(1–7) 4 weeks, and endothelial cell proliferation in these ROIs of the ipsilateral hemisphere (C) and the contralateral hemisphere (D) was evaluated by double immunofluorescence staining with CD31 and EdU. Bars: 50 μm. Data shown are means ± SD; n = 6 per group; *P < 0.05 significantly different from vehicle-treated rats.

Figure 2

Ang-(1–7) infusion activated eNOS and increased levels of NO and VEGF in the ipsilateral hemisphere. Rats were infused with Ang-(1–7) or vehicle for 4 weeks. The ratios of p-eNOS (Ser¹¹⁷⁷)/eNOS as well as the protein levels of nNOS and iNOS in the ipsilateral hemisphere (A) and the contralateral hemisphere (B) were evaluated by Western blot. (C, D) The levels of NO and VEGF in the ipsilateral hemisphere and the contralateral hemisphere. Data shown are means ± SD; n = 6 per group; *P < 0.05 significantly different from vehicle-treated rats.

Figure 3

Effects of A-779 and L-NIO on Ang-(1–7)-induced angiogenesis. Rats were co-treated with Ang-(1–7) and Mas antagonist A-779 or eNOS inhibitor L-NIO for 4 weeks. (A) The p-eNOS (Ser¹¹⁷⁷)/eNOS ratio in the ipsilateral hemisphere after co-treatment with A-779 or L-NIO. (B) The levels of NO and VEGF in the ipsilateral hemisphere after co-treatment with A-779 or L-NIO. (C) Capillary density in cerebral cortex of the ipsilateral hemisphere after co-treatment with A-779 or L-NIO was assessed by CD31 immunofluorescence staining. (D) Endothelial cell proliferation in cerebral cortex of the ipsilateral hemisphere after co-treatment with A-779 or L-NIO was evaluated by double immunofluorescence staining with CD31 and EdU. Data shown are means ± SD; n = 6 per group; *P < 0.05 significantly different from Ang-(1–7)-treated rats.

Figure 4

Ang-(1–7) infusion attenuated the reduction of rCBF after pMCAO. Rats were co-treated with Ang-(1–7) and Mas receptor antagonist A-779, eNOS inhibitor L-NIO or an anti-angiogenic compound endostatin (Endo) for 4 weeks, and were subjected to pMCAO. CBF was measured in the peripheral region (A) and core region (B) of the MCA territory before and after pMCAO. Data are expressed as a percentage of basal CBF in vehicle-treated rats. Data shown are means ± SD; n = 10-11 per group; *P < 0.05 significantly different from vehicle-treated rats. #P < 0.05 significantly different from Ang-(1–7)-treated rats.

Figure 5

Ang-(1–7) infusion reduced infarct volume and neurological deficits after pMCAO. Rats were co-treated with Ang-(1–7) and Mas antagonist A-779, eNOS inhibitor L-NIO or an anti-angiogenic compound endostatin (Endo) for 4 weeks, and were subjected to pMCAO. (A) Representative TTC images showing the mean infarct volume in each group at 24 h after pMCAO. (B) Infarct volume in each group at 24 h after pMCAO. (C) The distribution of neurological deficit score in each group at 24 h after pMCAO. With the exception of neurological deficit, Data shown are means ± SD; n = 10-11 per group; *P < 0.05 significantly different from vehicle-treated rats. #P < 0.05 significantly different from Ang-(1–7)-treated rats.

Figure 6

Diagram illustrating the mechanisms by which brain Ang-(1–7) promoted angiogenesis and induced tolerance against focal cerebral ischaemia.