ACE2 Attenuates Epithelial-Mesenchymal Transition in MLE-12 Cells Induced by Silica

Abstract

Purpose

The role of angiotensin-converting enzyme 2 (ACE2) in silicosis remains unknown, although previous studies have suggested that ACE2 may be beneficial. We, therefore, investigated the effect of ACE2 on silicosis, particularly with regard to its role in regulating the epithelial-mesenchymal transition (EMT) induced by silica, with the aim to uncover a new potential target for the treatment of pulmonary fibrosis.

Materials and Methods

We employed wild-type mice treated with diminazene aceturate (DIZE, an ACE2 activator, 15 mg/kg/day for 4 weeks), hACE2-transgenic mice (overexpress the ACE2 gene), and the mouse lung type II epithelial cell line treated with DIZE (10⁻⁷ M for 48 h) or angiotensin-(1–7) [Ang-(1–7)] (10⁻⁴ M for 48 h), following induced fibrotic responses to determine the protective potential of ACE2. Silicosis models were established by orotracheal instillation of SiO₂ (2.5 mg/mouse). Immunostaining was used to determine α-smooth muscle actin (α-SMA) expression. The activities of angiotensin-converting enzyme (ACE) and ACE2 and the levels of angiotensin II (Ang II) and Ang-(1-7) were detected by enzyme-linked immunosorbent assay. The mRNA expression of ACE and ACE2, and protein expression of the renin-angiotensin system (RAS) components and EMT indicators were studied by qRT-PCR and Western blot, respectively.

Results

DIZE treatment and overexpression of ACE2 markedly inhibited the formation of silica-induced lung fibrosis and increased the level of E-cadherin, with concomitant downregulation of pro-collagen, vimentin, and α-SMA via RAS signaling. Furthermore, DIZE and Ang-(1–7) attenuated the EMT and collagen deposition induced by silica in MLE-12 cells. Moreover, these effects were abrogated by MLN-4760 (a specific ACE2 inhibitor) and A779 (a specific Mas receptor blocker).

Conclusion

The overexpression of ACE2 and treatment with DIZE can ameliorate EMT in silicotic mice via activation of the ACE2-Ang-(1–7)-Mas receptor axis, and these changes are accompanied by suppression of the ACE–Ang II–AT1 receptor axis.

Keywords:

• angiotensin-converting enzyme 2
• angiotensin-converting enzyme
• silicosis fibrosis
• diminazene aceturate
• renin–angiotensin system
• transgenic mice

Acknowledgments

We thank Natasha Beeton-Kempen, PhD., from Liwen Bianji, Edanz Editing China for editing the English text of a draft of this manuscript.

Abbreviations

SiO₂, silicon dioxide; RAS, renin–angiotensin system; ACE, angiotensin-converting enzyme; ACE2, angiotensin-converting enzyme 2; Ang II, angiotensin II; Ang-(1–7), angiotensin-(1-7); AT1, angiotensin II receptor 1; DIZE, diminazene aceturate; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix.

Author Contributions

Shumin Li and Hong Xu designed the study. Shumin Li, Yaqian Li, Zhongqiu Wei, Yi Yang, Fuyu Jin, Min Zhang, Chen Wang, Wenxiong Song, Jingchen Huo and Jingyuan Zhao carried out the experimental work, analyzed the data, and drafted the manuscript. Fang Yang and Xiuhong Yang participated in the design of the study, critically reviewed the manuscript, and provided intellectual input. All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.