Ang-(1-7)/ MAS1 receptor axis inhibits allergic airway inflammation via blockade of Src-mediated EGFR transactivation in a murine model of asthma

The angiotensin-(1�7) [Ang-(1�7)]/MAS1 receptor signaling axis is a key endogenous anti-inflammatory signaling pathway. However, the mechanisms by which its mediates the anti-inflammatory effects are not completely understood. Using an allergic murine model of asthma, we investigated whether Ang-1(1�7)/MAS1 receptor axis a): inhibits allergic inflammation via modulation of Src-dependent transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling effectors such as ERK1/2, and b): directly inhibits neutrophil and/or eosinophil chemotaxis ex vivo. Ovalbumin (OVA)-induced allergic inflammation resulted in increased phosphorylation of Src kinase, EGFR, and ERK1/2. In addition, OVA challenge increased airway cellular influx, perivascular and peribronchial inflammation, fibrosis, goblet cell hyper/metaplasia and airway hyperresponsiveness (AHR). Treatment with Ang-(1�7) inhibited phosphorylation of Src kinase, EGFR, ERK1/2, the cellular and histopathological changes and AHR. Ang-(1�7) treatment also inhibited neutrophil and eosinophil chemotaxis ex vivo. These changes were reversed following pretreatment with A779. These data show that the anti-inflammatory actions of Ang-(1�7)/ MAS1 receptor axis are mediated, at least in part, via inhibition of Src-dependent transactivation of EGFR and downstream signaling molecules such as ERK1/2. This study therefore shows that inhibition of the Src/EGRF/ERK1/2 dependent signaling pathway is one of the mechanisms by which the Ang-(1�7)/ MAS1 receptor axis mediates it anti-inflammatory effects in diseases such as asthma. - 2019 El-Hashim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.This study was supported by Kuwait University Research Sector - grant # PT01/12. Parts of this work were supported by the research grant # SRUL02/12 to the Research Unit for Genomics, Proteomics and Cellomics Studies through the Research Core Facility. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors are thankful to the support of Mr. Sunny Ojoko and Mr. Hecktor Velasco from the Animal Resources Center of Health Sciences Center. The authors are also very grateful to Dr. Ananthalakshmi KV, Ms. Sowmya Balakrishnan and Ms. Preethi Tobin for their excellent work with the immunofluorescence studies and histological studies.Scopu

Effect of Ang-(1–7) (0.3 mg/kg; i.p) and A779 on ovalbumin-induced change in total BALF cell count, eosinophils, lymphocytes, neutrophils and macrophage.

Treatment with A779 significantly attenuated the Ang-(1–7)-mediated inhibition of the ovalbumin-induced increase in total cell influx, eosinophils, lymphocytes and neutrophils in the airways. Data are expressed as mean ± SEM (n = 8–14). *P #P < 0.05 versus time-matched ovalbumin-challenged mice.</p

Immunofluorescent (Alexa Fluor) detection of phosphorylated Src shown on the upper panels are overlaid with DAPI stain on the lower panel to show lung tissue architecture.

Lung sections were taken from different treatment groups and immunostained for phosphorylated Src (Fig 1(b–f)). Negative control (a); PBS/Veh (b); OVA/Veh (c); OVA/Ang-(1–7) (d); OVA/Ang-(1–7) + A779 (e) and OVA/Dex (f). PBS treated mice showed minimal pSrc (b). OVA challenge resulted in a significant increase in pSrc and this was inhibited following treatment with Ang-(1–7) (0.3 mg/kg) (c, d and g) and was comparable to the dexamethasone treated animals (f and g). Treatment with A779 inhibited the Ang-(1–7) (0.3 mg/kg)–induced decrease in pSrc (e and g). Quantitative assessment of fluorescence intensity of p-Src (Fig 1(g)) (arbitrary units). Data are expressed as mean ± SEM (n = 4–8). *P #P ¥ P < 0.05 versus time-matched OVA/Ang-(1–7) treated animals. Panel (h) is a higher magnification (x40) (x40) of the OVA challenged group to better show the localization of the stain.</p

Immunofluorescent (Alexa Fluor) detection of phosphorylated ERK1/2 shown on the upper panels are overlaid with DAPI stain on the lower panel to show lung tissue architecture.

Lung sections were taken from different treatment groups and immunostained for ERK1/2 (Fig (1b–1f)). Negative control (a); PBS/Veh (b); OVA/Veh (c); OVA/Ang-(1–7) (d); OVA/Ang-(1–7) + A779 (e) and OVA/Dex (f) (x 20 magnification). PBS treated mice showed minimal pERK1. OVA challenge resulted in a significant increase in ERK1/2 and this was inhibited following treatment with Ang-(1–7) (0.3 mg/kg) (c, d and g) and was comparable to the dexamethasone treated animals (f and g). Treatment with A779 inhibited the Ang-(1–7) (0.3 mg/kg)–induced decrease in ERK1/2 (e and g). Quantitative assessment of fluorescence intensity of ERK1/2 (Fig 1(g)) (arbitrary units). Data are expressed as mean ± SEM (n = 5–11). *P #P ¥ P < 0.05 versus time-matched OVA/Ang-(1–7) treated animals. Panel (h) is a higher magnification (x40) of the OVA challenged group to better show the localization of the stain.</p

Western blot analysis of pSrc, pEGFR and p-ERK1/2 protein levels from lungs of PBS challenged mice pretreated with vehicle (PBS), ovalbumin challenged pretreated with vehicle (OVA), ovalbumin challenged pretreated with Ang-(1–7) (Ang-(1–7)), ovalbumin mice challenged pretreated with A779 + Ang-(1–7) (A779) and ovalbumin challenged pretreated with dexamethasone (DEX).

The blots are representative of two similar but independent experiments (n = 3). Graphs a, b and c are densitometric quantification showing relative levels of p-Src, p-EGFR and p-ERK1/2, respectively (normalized to β-actin) of the shown blot.</p

Immunofluorescent (Alexa Fluor) detection of phosphorylated EGFR shown on the upper panels are overlaid with DAPI stain on the lower panel to show lung tissue architecture.

Lung sections were taken from different treatment groups and immunostained for pEGFR (Fig (1b–1f)). Negative control (a); PBS/Veh (b); OVA/Veh (c); OVA/Ang-(1–7) (d); OVA/Ang-(1–7) + A779 (e) and OVA/Dex (f). PBS treated mice showed minimal pEGFR (b). (a and b). OVA challenge resulted in a significant increase in pEGFR and this was inhibited following treatment with Ang-(1–7) (0.3 mg/kg) (c, d and g) and was comparable to the dexamethasone treated animals (f and g). Treatment with A779 inhibited the Ang-(1–7) (0.3 mg/kg)–induced decrease in pEGFR (e and g). Quantitative assessment of fluorescence intensity of pEGFR (Fig 1(g)) (arbitrary units). Data are expressed as mean ± SEM (n = 6–8). *P #P ¥ P < 0.05 versus time-matched OVA/Ang-(1–7) treated animals. Panel (h) is a higher magnification (x40) of the OVA challenged group to better show the localization of the stain.</p

Effect of Ang-(1–7) (0.3 mg/kg), Ang-(1–7) (0.3 mg/kg) and A779 (1 mg/kg), and dexamethasone (1 mg/kg) on ovalbumin-induced AHR to inhaled methacholine.

Lung function measurements were done 24 h after the last challenge. OVA challenged mice had significant AHR compared with the PBS/Veh group. Treatment with both Ang-(1–7) (0.3 mg/kg) and dexamethasone (1 mg/kg) both significantly reduced the OVA-induced AHR. Data are expressed as mean ± SEM (n = 12–19).</p