Mechanical ventilation augments bleomycin-induced epithelial–mesenchymal transition through the Src pathway

Abstract

Mechanical ventilation used in patients with acute respiratory distress syndrome (ARDS) can damage pulmonary epithelial cells by producing inflammatory cytokines and depositing excess collagen. Src participates in plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor-b1(TGF-b1) production during the fibroproliferative phase of ARDS, which involves a process of epithelial-mesenchymal transition (EMT). The mechanisms regulating interactions between mechanical ventilation and EMT are unclear. We hypothesized that EMT induced by high-tidal volume (V T ) mechanical stretch-augmented lung inflammation occurs through upregulation of the Src pathway. Five days after administering bleomycin to simulate acute lung injury (ALI), male C57BL/6 mice, either wild-type or Src-deficient, aged 3 months, weighing between 25 and 30 g, were exposed to low-V T (6 ml/kg) or high-V T (30 ml/kg) mechanical ventilation with room air for 1-5 h. Nonventilated mice were used as control subjects. We observed that high-V T mechanical ventilation increased microvascular permeability, PAI-1 and TGF-b1 protein levels, Masson's trichrome staining, extracellular collagen levels, collagen gene expression, fibroblast accumulation, positive staining of a-smooth muscle actin and type I collagen, activation of Src signaling and epithelial apoptotic cell death in wild-type mice (Po0.05). Decreased staining of the epithelial marker, Zonula occludents-1, was also observed. Mechanical stretch-augmented EMT and epithelial apoptosis were attenuated in Src-deficient mice and pharmacological inhibition of Src activity by PP2 (Po0.05). Our data suggest that high-V T mechanical ventilation-augmented EMT after bleomycin-induced ALI partially depends on the Src pathway. Acute respiratory distress syndrome (ARDS) is a disorder of acute respiratory failure and manifests as non-cardiogenic pulmonary edema, respiratory distress and hypoxemia. 1,2 Mechanical ventilation with high-tidal volume (V T ) causes severe lung injury (ventilator-induced lung injury (VILI)) characterized by an initial inhomogeneous inflammatory reaction or epithelial injury that is followed by a fibroproliferative phase with fibroblast proliferation. 3-8 Epithelialmesenchymal transition (EMT) is the differentiation of epithelial cells into myofibroblast-like cells, which contributes to the fibroproliferative phase. 3-8 Upregulating Src, the plasminogen activator inhibitor-1 (PAI-1), and the transforming growth factor-b1(TGF-b1) has recently been demonstrated to regulate EMT. 9-14 However, the mechanisms regulating the interactions between mechanical ventilation and EMT remain unclear.PAI-1, a member of the serine protease inhibitor (serpin) gene family, rapidly inhibits both the urokinase-type plasminogen activator and the tissue-type plasminogen activator (tPA). 15 Mice with homozygous deletion of PAI-1 were relatively protected from bleomycin-induced pulmonary fibrogenesis, whereas overexpression of PAI-1 enhanced pulmonary fibrogenesis. 9 As a primary profibrogenic cytokine...

Nintedanib reduces ventilation‐augmented bleomycin‐induced epithelial–mesenchymal transition and lung fibrosis through suppression of the Src pathway

“…Wild‐type or Src‐deficient C57BL/6 mice, aged between 6 and 8 weeks, weighing between 20 and 25 g, were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and National Laboratory Animal Center (Taipei, Taiwan), as described in our previous study . We performed the experiments in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals.…”

“…We used our established mouse model of VILI, as previously described . In brief, a 20‐gauge angiocatheter was introduced into the tracheotomy orifice of mice and general anaesthesia was maintained by regular intraperitoneal administration of zoletil 50 (5 mg/kg) and xylazine (5 mg/kg) at the beginning of experiment and every 30 min.…”

“…Nintedanib was administered once daily through gavage at 30, 60 and 100 mg/kg for 5 days before MV (Boehringer Ingelheim, Biberach, Germany) . The mice received a single dosage of 0.075 units of bleomycin in 100 μl of sterile normal saline solution intratracheally (Sigma‐Aldrich, St. Louis, MO, USA) and were ventilated 5 days after the administration of bleomycin .…”

“…It is a non‐receptor tyrosine kinase that is crucial for intracellular signal transduction related to cell proliferation, migration, differentiation, adhesion and apoptotic cell death . A previous murine study demonstrated that MV can augment bleomycin‐induced EMT through the Src pathway . The profibrotic effect of Src kinases could be an attractive target in the management of pulmonary fibrosis by using current novel tyrosine kinase inhibitors .…”

Src promotes EGF‐induced epithelial‐to‐mesenchymal transition and migration in gastric cancer cells by upregulating ZEB1 and ZEB2 through AKT

“…Src, the intracellular non-receptor tyrosine kinase, is implicated in multiple signaling pathways regulating cellular proliferation, angiogenesis, cancer invasion and metastasis, and bone metabolism (Aleshin and Finn, 2010). Furthermore, Src has been reported to be involved in EMT induced under various conditions (Nagaharu et al, 2011;Li et al, 2014a;Moon et al, 2014;Martinez-Revollar et al, 2015). However, it is still unclear whether Src regulates EGFinduced EMT in gastric cancer cells.…”