Stretch‐induced activation of Hippo signaling in lung microvascular endothelial cells – a novel mechanism of overventilation‐induced pulmonary fibrosis

Abstract

Mechanical ventilation is a mainstay of current therapy for patients with acute respiratory distress syndrome (ARDS). Yet despite its benefits, mechanical ventilation can also cause ventilator induced lung injury (VILI) that may ultimately progress to pulmonary fibrosis. We aimed to elucidate cellular mechanisms by which mechanical ventilation triggers pulmonary fibrosis. Specifically, we focused on the mechanosensitive transcriptional co‐factor TAZ, a key player in the HIPPO‐signaling pathway involved in mechanosensation. In the HIPPO pathway, phosphorylation of Large Tumor Suppressor Kinases (LATS) regulates the phosphorylation state of TAZ and its translocation to the nucleus. Despite its emerging relevance in tissue fibrosis, TAZ activation, its underlying mechanisms and downstream profibrotic effects upon mechanical overventilation or its in vitro correlate cyclic stretch have not been elucidated.ObjectivesTo examine the effect of mechanical stretch on the nuclear translocation of TAZ in endothelial cells, and to identify underlying regulatory mechanisms effects both in vitro and in vivo.MethodsIn vitro: human pulmonary microvascular endothelial cells (HPMEC) were subjected to stretch regimens at 5%, mimicking low tidal volume ventilation, or 18% mimicking high tidal volume ventilation at 0.25 Hz for 1, 3, 6, or 12h on a Flexcell Tension System and compared to static control. In vivo: To mimic ARDS in mice, Endothelial TAZ‐deficient and corresponding control were challenged with HCl (pH 1.0; 3 mL/kg), recovered for 3 days, ventilated for 2 hours at VT of 20ml/kg and PEEP of 2 cmH2O; and compared to unventilated controls. Evidence of fibrosis was detected histologically two weeks after ventilation.ResultsIn vitro: Mechanical stretch induced the translocation of TAZ from the cytosol to the nucleus in a dose‐ and time‐dependent manner. Specifically, the nuclear to cytoplasmic ratio of TAZ increased after 3h of stretch at 18% compared to the control whereas stretching cells at 5% increased TAZ nuclear localization after 6h. Stretch‐induced TAZ translocation was blocked by the inhibition of Aurora kinases with SNS 314 mesylate (10 μMol/L). Consistently, inhibition of Aurora kinases decreased LATS2 phosphorylation at Ser83, a modulatory site of its activity. In vivo: Histological analysis of lung tissue using Masson’s trichrome and H&E stain showed marked peribronchiolar and perivascular collagen deposition as well as bronchial wall thickening due to extensive infiltration of inflammatory cells in ventilated control, yet not in endothelial TAZ‐deficient mice.ConclusionThese data suggest that mechanical stretch activates TAZ nuclear translocation, at least in part, via an Aurora kinase and LATS2‐dependent mechanism. Knocking down TAZ in endothelial cells protected mice from developing pulmonary fibrosis following HCL‐overventilation. Interventions targeted at the Hippo‐signaling pathway and its upstream regulation may present potential strategies to counteract the detrimental effects of mechanical overventilation.Support or Funding InformationThis work was supported by the Canadian Institutes of Health Research.