Endothelin-converting enzyme-1 gene ablation attenuates pulmonary fibrosis via CGRP-cAMP/EPAC1 pathway

Endothelin-1 (ET-1) has been shown to be involved in human pulmonary fibrosis. However, recent clinical trials targeting the ET-1 pathway with ET-1 receptor antagonists failed to achieve beneficial outcomes. Another strategy opposing the actions of ET-1 involves the inhibition of endothelin-converting enzyme-1 (ECE-1). We hypothesize that ECE-1 inhibition exerts beneficial effects on pulmonary fibrosis. Pulmonary fibrosis was induced by instilling bleomycin intratracheally into ECE-1 heterozygous knockout mice (ECE-1(+/-)) and their wild-type control mice (ECE-1(+/+)). Lung inflammation and fibrosis were assessed on Days 7, 14, and 28 after bleomycin instillation. The activity of ECE-1 and the concentrations of its related peptides, ET-1, bradykinin, atrial natriuretic peptide (ANP), and calcitonin gene-related peptide (CGRP), were determined. ECE-1(+/-) mice demonstrated less lung inflammation and limited fibrosis compared with control mice. ECE-1 activity was half-reduced in ECE-1(+/-) mice, and this activity also altered ET-1 and CGRP concentrations, but not concentrations of bradykinin and ANP. ET-1 concentrations were found to be lower in ECE-1(+/-) mice after the development of fibrosis, in contrast to the unaltered concentrations during inflammation. Reduced ECE-1 activity resulted in higher CGRP concentrations, which altered the pathological functionality of the lung, indicating the activation of the CGRP pathway involving cyclic adenosine monophosphate (cAMP)/exchange protein directly activated by cAMP and cAMP/protein kinase A in ECE-1(+/-) mice. Bleomycin instillation on Day 14 induced the accumulation of M2 macrophages expressing CGRP receptors in ECE-1(+/-) mice. Our results emphasize that the in vivo ECE-1-mediated degradation of CGRP promotes the transition from lung inflammation to fibrosis. Further, our study identified M2 macrophages as the target cells of CGRP action during this transition.