Angiotensin-(1-7) counteracts the effects of Ang II on vascular smooth muscle cells, vascular remodeling and hemorrhagic stroke: Role of the NFкB inflammatory pathway

Abstract

Angiotensin (Ang)-(1-7) is a potential vasoprotective peptide. In the present study, we investigated its counteractive effects to Ang II on vascular smooth muscle cells (VSMCs) and intracerebral hemorrhagic stroke (ICH) through inflammatory mechanism. In in vitro experiments, human brain VSMCs (HBVSMCs) were treated with vehicle, Ang II, Ang II+Ang-(1-7), Ang II+A-779 or Ang II+Ang-(1-7)+A-779 (Mas receptor antagonist). HBVSMC proliferation, migration and apoptosis were determined by methyl thiazolyltetrazolium, wound healing assay and flow cytometry, respectively. In in vivo experiments, C57BL/6 mice were divided into vehicle, Ang II, Ang II+Ang-(1-7), Ang II+A-779 or Ang II+Ang-(1-7)+A-779 groups before they were subjected to collagenase-induced ICH or sham surgery. Hemorrhage volume and middle cerebral artery (MCA) remodeling were determined by histological analyses. Levels of NFκB, inhibitor of κBα (IκBα), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interleukin (IL-8) were measured by western blot or ELISA. We found that 1) Ang II increased HBVSMC migration, proliferation and apoptosis, and increased the blood pressure (BP), neurological deficit score, MCA remodeling and hemorrhage volume in ICH mice. 2) Ang-(1-7) counteracted these effects of Ang II, which was independent of BP, with the down-regulation of NFκB, up-regulation of IκBα, and decreased levels of TNF-α, MCP-1 and IL-8. 3) The beneficial effects of Ang-(1-7) could be abolished by A-779. In conclusion, Ang-(1-7) counteracts the effects of Ang II on ICH via modulating NFκB inflammation pathway in HBVSMCs and cerebral microvessels.

Keywords: Ang II; Ang-(1–7); Hemorrhagic stroke; NFκB; VSMCs.

Fig. 1. Experimental procedures and time flow chart

HBVSMCs: human brain smooth muscle cells; Veh: vehicle; Ang II: angiotensin II; NFκB: nuclear factor-KappaB; MCA: middle cerebral artery; ICH: intracerebral hemorrhage. TNF-α: tumor necrosis factor; MCP-1: monocyte chemoattractant protein 1; IL-8: interleukin-8; NDS: neurological deficit scores.

Fig. 2. The effects of Ang-(1-7) on HBVSMC apoptosis, proliferation and migration

A, Ang-(1-7) protects HBVSMCs from Ang II-induced apoptosis. ⁺⁺P< 0.01 vs. 0 M Ang (1-7), n=6/group. B, Ang-(1-7) decreased the Ang II-induced increased in HBVSMC proliferation. C, Representative images of migration abilities of HBVSMCs. Scale bars: 200 μm. D, Ang-(1-7) decreased the Ang II-induced increased in HBVSMC migration. **P< 0.01 vs. veh; ⁺⁺P< 0.01 vs. Ang II, ^#P< 0.05, ^##P< 0.01 vs. Ang II + Ang-(1-7), n=6/group.

Fig. 3. The effects of Ang-(1-7) on regulating gene expressions and cytokines release of HBVSMCs

Ang II induced the up-regulation of NFκB (A) and down-regulation of IκBα (B) in HBVSMCs. In addition, Ang II induced the release of TNF-α (C), MCP-1 (D) and IL-8 (E) from HBVSMCs. Ang-(1-7) counteracted these effects of Ang II, which could be aborted by A-779. **P< 0.01 vs. veh; ⁺⁺P< 0.01 vs. Ang II, ^##P< 0.01 vs. Ang II + Ang-(1-7), n=6/group. TNF-α: tumor necrosis factor; MCP-1: monocyte chemoattractant protein 1; IL-8: interleukin-8.

Fig. 4. The effects of Ang-(1-7) on MAP and MCA remodeling

A, Representative pictures of HE staining for MCA remodeling. Scale bars: 50 μm. B, Ang II infusion increased MAP. Ang-(1-7) infusion had no effect on MAP. C, Ang II infusion induced MCA remodeling in mice, while Ang-(1-7) could alleviate Ang II-induced MCA remodeling. Mas receptor (A-779) could block the effects of Ang-(1-7). **P< 0.01 vs. veh; ⁺⁺P< 0.01 vs. Ang II, ^##P< 0.01 vs. Ang II + Ang-(1-7), n=10/group. MAP: mean arterial pressure; MCA: middle cerebral artery.

Fig. 5. The effects of Ang-(1-7) on hemorrhage volume in ICH

A, Representative pictures of hemorrhage volume in different treatment groups. Scale bars: 2 mm. B, Ang II infusion enlarged the hemorrhage volume, while Ang-(1-7) infusion decreased the hemorrhage volume. C, Neurological deficits were scored in ICH mice. Ang II infusion exaggerated the neurological deficits, while Ang-(1-7) infusion alleviated the neurological deficits. A-779 could abolish the protective effects of Ang-(1-7). ^&&P< 0.01 vs. sham; **P< 0.01 vs. veh; ⁺⁺P< 0.01 vs. Ang II, ^##P< 0.01 vs. Ang II + Ang-(1-7), n=10/group.

Fig. 6. The effects of Ang-(1-7) on regulating gene expressions and cytokine levels in cerebral microvessels

The protein expression of NFκB (A) in cerebral microvessels was up-regulated by Ang II infusion, while the expression of IκBα (B) was down-regulated. The levels of TNF-α (C), MCP-1 (D) and IL-8 (E) in the cerebral microvessels were increased by Ang II infusion. Ang-(1-7) blocked all these effects of Ang II. A-779 treatment reversed the effects of Ang-(1-7). ^&P< 0.05, ^&&P< 0.01 vs. sham; **P< 0.01 vs. veh; ⁺P< 0.05, ⁺⁺P< 0.01 vs. Ang II, ^##P< 0.01 vs. Ang II + Ang-(1-7), n=10/group.