Angiotensin-(1–7) alleviates acute lung injury by activating the Mas receptor in neutrophil

Figures

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  Figure 1. Effect of Ang-(1–7) and its receptor on lung pathological changes in acid- and ventilation-induced ALI mice. Group data showing the effects of Ang-(1–7) (6 nmol/kg) when administered either alone or in combination with the Ang-(1–7) receptor antagonist A779 (12 nmol/kg) concomitantly with the induction of acid- and ventilation-induced lung injury on the wet-to-dry lung weight ratio (A), lung MPO activity (B), and histological features of lung injury on a quantitative scale from 0 (no injury) to 1 (C). Histological features of lung injury are shown by representative histological micrographs by H&E staining (D). Data are mean ± SEM from n=5 mice per group. *, P<0.05 versus healthy control mice; †, P<0.05 versus HCl and over ventilation; ‡, P<0.05 versus HCl and over ventilation + Ang-(1–7) i.v.; #, P<0.05 versus HCl and over ventilation + Ang-(1–7) i.t.. ALI, acute lung injury; MPO, myeloperoxidase; SEM, standard error of mean; HCl, hydrochloric; i.v., intravenous injection; i.t., intratracheal instillation; H&E, hematoxylin and eosin.

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  Figure 2. Effects of Ang-(1–7) and its receptor on the lung inflammatory cytokine response of ALI. Group data showing the effects of Ang-(1–7) (6 nmol/kg) when administered either alone or in combination with the Ang-(1–7) receptor antagonist A779 (12 nmol/kg) concomitantly with the induction of acid- and ventilation-induced lung injury on the concentration of TNF-α (A), IL-1ß (B), IL-6 (C), and IL-10 (D). The data are expressed as the mean ± SEM from n=5 mice per group. *, P<0.05 versus healthy control mice; †, P<0.05 versus HCl and over ventilation; ‡, P<0.05 versus HCl and over ventilation + Ang-(1–7) i.v.; #, P<0.05 versus HCl and over ventilation + Ang-(1–7) i.t.. ALI, acute lung injury; TNF-α, tumor necrosis factor-α; IL, interleukin; SEM, standard error of mean; HCl, hydrochloric; i.v., intravenous injection; i.t., intratracheal instillation.

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  Figure 3. Mas receptor expression in human neutrophils. Mas receptor expression was detected in human neutrophils by quantitative RT-PCR (A), and western blot analyses (B). Human HUVECs were used as a control cell line. The data are presented as the mean ± SEM of 3 independent experiments. HUVEC, human umbilical vein endothelial cell; RT-PCR, real-time polymerase chain reaction; SEM, standard error of mean.

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  Figure 4. Ang-(1–7) attenuates respiratory burst, adhesion, and transmigration of neutrophils. Group data showing neutrophil ROS production (A), neutrophil adhesion to the HPMECs (B), and neutrophil chemotaxis across the monolayer of the HPMECs (C) when Ang-(1–7) (0.1 µmol/L) was administered either alone or in combination with the Ang-(1–7) receptor antagonist A779 (10 µmol/L) concomitantly in response to stimulation with PAF (10 mmol/L) in neutrophils. The data are presented as the mean ± SEM from n=3 per group. *, P<0.05 versus PAF control; #, P<0.05 versus PAF + Ang-(1–7). ROS, reactive oxygen species; HPMEC, human pulmonary microvascular endothelial cell; PAF, platelet-activating factor; SEM, standard error of mean.

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  Figure 5. Neutrophil-depleting antibody anti-Ly6G reduces neutrophil recruitment in lungs. Anti-Ly6G antibody (clone 1A8, 200 µg) was administered intraperitoneally either alone or in combination with the Ang-(1–7) receptor antagonist A779 (12 nmol/kg) concomitantly with the induction of the LPS-induced ALI. Neutrophil depletion was confirmed by MPO immunofluorescence staining as shown by representative images in (A) and the percentage of MPO positive cells in (B). The data are presented as the mean ± SEM from n=5 mice per group. *, P<0.05 versus healthy control mice; †, P<0.05 versus LPS; #, P>0.05 versus vs. Anti-Ly6G + LPS. LPS, lipopolysaccharide; ALI, acute lung injury; MPO, myeloperoxidase; SEM, standard error of mean.

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  Figure 6. Depletion of neutrophils attenuates ALI in an animal model. Group data showing the effects of the anti-Ly6G antibody (clone 1A8, 200 µg, intraperitoneally) when administered either alone or in combination with the Ang-(1–7) receptor antagonist A779 (12 nmol/kg) concomitantly with the induction of the LPS-induced ALI on BALF protein concentration (A), and histological features of lung injury on a quantitative scale from 0 (no injury) to 1 (B). The histological features of lung injury are shown by representative histological micrographs by H&E staining (C). The data are presented as the mean ± SEM from n=5 mice per group. *, P<0.05 versus healthy control mice; †, P<0.05 versus LPS; #, P>0.05 versus LPS; ‡, P<0.05 versus anti-Ly6G + LPS. LPS, lipopolysaccharide; ALI, acute lung injury; BALF, bronchoalveolar lavage fluid; SEM, standard error of mean.