Mechanosensitive ion channel Piezo1 mediates mechanical ventilation-exacerbated ARDS-associated pulmonary fibrosis

Abstract

Introduction: Pulmonary fibrosis is a major cause of the poor prognosis of acute respiratory distress syndrome (ARDS). While mechanical ventilation (MV) is an indispensable life-saving intervention for ARDS, it may cause the remodeling process in lung epithelial cells to become disorganized and exacerbate ARDS-associated pulmonary fibrosis. Piezo1 is a mechanosensitive ion channel that is known to play a role in regulating diverse physiological processes, but whether Piezo1 is necessary for MV-exacerbated ARDS-associated pulmonary fibrosis remains unknown.

Objectives: This study aimed to explore the role of Piezo1 in MV-exacerbated ARDS-associated pulmonary fibrosis.

Methods: Human lung epithelial cells were stimulated with hydrochloric acid (HCl) followed by mechanical stretch for 48 h. A two-hitmodel of MV afteracidaspiration-inducedlunginjuryin mice was used. Mice were sacrificed after 14 days of MV. Pharmacological inhibition and knockout of Piezo1 were used to delineate the role of Piezo1 in MV-exacerbated ARDS-associated pulmonary fibrosis. In some experiments, ATP or the ATP-hydrolyzing enzyme apyrase was administered.

Results: The stimulation of human lung epithelial cells to HCl resulted in phenotypes of epithelial-mesenchymal transition (EMT), which were enhanced by mechanical stretching. MV exacerbated pulmonary fibrosis in mice exposed to HCl. Pharmacologicalinhibitionorknockout of Piezo1 attenuated the MV-exacerbated EMT process and lung fibrosis in vivo and in vitro. Mechanistically, the observed effects were mediated by Piezo1-dependent Ca²⁺ influx and ATP release in lung epithelial cells.

Conclusions: Our findings identify a key role for Piezo1 in MV-exacerbated ARDS-associated pulmonary fibrosis that is mediated by increased ATP release in lung epithelial cells. Inhibiting Piezo1 may constitute a novelstrategyfor the treatment of MV-exacerbated ARDS-associated pulmonary fibrosis.

Keywords: Acute respiratory distress syndrome; Mechanical ventilation; Piezo1; Pulmonary fibrosis.

Graphical abstract

Fig. 1

MV exacerbated pulmonary fibrosis and EMT in vitro and in vivo.(a-d) BEAS-2B cells were exposed to mechanical stretch (20 % elongation) for 48 h with or without HCl (pH 4) priming for 30 min. n = 6 each, *P < 0.05, **P < 0.01 vs. the Static + PBS group; ^#P < 0.05 vs. the Static + HCl group. (a) Schematic of the in vitro models used. (b) The expression of E-cadherin, cytokeratin-8, vimentin and α-SMA in BEAS-2B cells was measured by Western blot analysis. (c and d) The expression of E-cadherin, cytokeratin-8 vimentin and α-SMA was confirmed by immunostaining. Bar = 75 µm. (e-m) Mice were exposed to MV for 2 h following intratracheal instillation of HCl (pH 1.2, 2 ml/kg). n = 6 each, *P < 0.05, **P < 0.01, vs. the PBS group; ^#P < 0.05, ^##P < 0.01 vs. the HCl group. (e) Schematic of the in vivo models used. (f-h) The lung injury score and lung fibrosis score were determined based on lung histological analysis with HE staining and Masson’s trichrome staining. Original magnification, ×200. Lung tissue levels of (i) hydroxyproline and (j) collagen-1 on day 14. (k) The protein expression of E-cadherin, cytokeratin-8, vimentin and SMA was measured by Western blot analysis. A representative image from three independent experiments is presented. (l and m) The expression of E-cadherin, cytokeratin-8, vimentin and SMA was confirmed by immunostaining. Bar = 40 µm.

Fig. 2

Mechanical stretch induced Piezo1 activation in bronchial airway epithelial cells.(a) The expression of Piezo1 in human lung epithelial cells (BEAS-2B) (bar = 75 µm) and the context of mouse lung tissue (bar = 40 µm). (b) qPCR analysis of known mammalian mechanosensory ion channels in unstimulated BEAS-2B cells. n = 3 each, *P < 0.05, **P < 0.01 vs. Piezo1. (c) Piezo1 protein expression in BEAS-2B cells exposed to mechanical stretch. n = 3 each, *P < 0.05 vs. Control. (d-g) BEAS-2B^WT cells and BEAS-2B^Piezo1-/- cells were exposed to mechanical stretch (20 % elongation) (d) or 25 μM Yoda1 (e-g) with HCl (pH 4) priming for 30 min (n = 6 each). BEAS-2B cells were primed with GsMTx4 (2.5 µM) or EGTA 30 min prior to mechanical stretch or Yoda1 treatment (n = 6 each). (d) The representative intracellular Ca²⁺ concentration after mechanical stretch (30 min) was determined using a flow cytometer. (e) The representative intracellular Ca²⁺ concentration after Yoda1 treatment (5 min) was determined using a flow cytometer. (f) Real-time dynamics of Ca²⁺ influx was assessed in live cells by flow cytometry. Cells were analyzed to establish the baseline. The addition of 25 μM Yoda1 resulted in rapid intracellular Ca²⁺ influx, as indicated by a shift in the Flu-3 mean fluorescence intensity (MFI). (g) A representative graph showing the shift in MFI over time. The red arrow indicates the point at which Yoda1 (25 µM) was added to the cells.

Fig. 3

Piezo1 knockdown inhibited MV-enhanced lung fibrosis and EMT in vitro and in vivo.(a -c) BEAS-2B^WT cells and BEAS-2B^Piezo1-/- cells were exposed to mechanical stretch (20 % elongation) for 48 h after HCl (pH 4) priming for 30 min. n = 6 each, *P < 0.05, **P < 0.01 vs. the BEAS-2B^WT + HCL + Stretch group. (a) The expression of E-cadherin, cytokeratin-8, vimentin and α-SMA was measured by Western blot analysis. (b and c) The expression of E-cadherin, cytokeratin-8, vimentin and SMA was confirmed by immunostaining. Bar = 75 µm. (d-k) Piezo1^CKO mice and Piezo1^F/F mice were exposed to MV for 2 h following intratracheal instillation of HCl (pH 1.2, 2 ml/kg). n = 6 each, *P < 0.05, **P < 0.01 vs. the Piezo1^F/F + HCl group; ^#P < 0.05, ^##P < 0.01 vs. the Piezo1^F/F + HCl + MV group. (d-f) The lung injury score and lung fibrosis score were determined based on lung histological analysis with HE staining and Masson’s trichrome staining. Original magnification, ×200. Lung tissue levels of (g) hydroxyproline and (h) collagen 1 on day 14. (i) The protein expression of E-cadherin, cytokeratin-8, vimentin and α-SMA was measured by Western blot analysis. (j and k) The expression of E-cadherin, cytokeratin-8, vimentin and SMA was confirmed by immunostaining. Bar = 40 µm.

Fig. 4

Mechanical stretch-induced ATP release from BEAS-2B cells depended on Piezo1.(a-c) BEAS-2B^WT cells and BEAS-2B^Piezo1-/- cells were exposed to 25 μM Yoda1 or (b) mechanical stretch (20 % elongation) after HCl (pH 4) stimulation for 30 min. BEAS-2B^WT cells were treated with GsMTx4 (2.5 µM) 30 min prior to mechanical stretch or Yoda1 administration. The cell culture medium was collected at different time points (0, 5, 15, 30 and 45 min) after mechanical stretch or Yoda1 administration. n = 6 each, *P < 0.05, **P < 0.01vs. 0 min. ^&P < 0.05, ^&&P < 0.01vs. the BEAS-2B^WT group. (a) The concentration of ATP in the cell culture medium after Yoda1 stimulation. (b) The concentration of ATP in the cell culture medium after mechanical stretch stimulation. (c) The concentration of LDH in the cell culture medium after mechanical stretch stimulation (45 min). (d) Male C57BL/6 mice were subjected to intratracheal instillation of HCl (pH 1.2, 2 ml/kg). After 24 h, the mice were administered GsMTx4 (270 µg/kg, intraperitoneally) and then subjected to MV for 2 h. BALF was collected after MV for different durations (0, 2, 6, 9 and 12 h). Then, the concentration of ATP was examined. n = 6 each, *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 min. &P < 0.05, &&P < 0.01, vs. the HCI + MV (Control) group. (e) Male C57BL/6 mice and Piezo1^CKO mice were subjected to intratracheal instillation of HCl (pH 1.2, 2 ml/kg). Twenty-four hours after HCl instillation, the mice were subjected to MV for 2 h. BALF was collected after 9 h of MV. Then, the concentration of ATP was examined. n = 6, **P < 0.01, vs. Piezo1^F/F. (f and g) BEAS-2B^WT cells and BEAS-2B^Piezo1-/- cells were exposed to mechanical stretch (20 % elongation) after HCl (pH 4) priming for 30 min. Cells were treated with BAPTA-AM or EGTA prior to mechanical stretch. The cell culture medium was collected after 45 min of mechanical stretch exposure. n = 6, *P < 0.05, **P < 0.01 vs. the control group. ^##P < 0.01 vs. the stretch group.

Fig. 5

eATP depletion suppressed MV-enhanced lung fibrosis and EMT in vitro and in vivo.(a- c) Human lung epithelial cells (BEAS-2B cells) underwent HCl (pH 4) stimulation for 30 min. After 4 h, the cells were then exposed to mechanical stretch (20 % elongation) for 48 h in the presence of the ATP-hydrolyzing enzyme apyrase (4 U/ml). n = 6 each, **P < 0.01 vs. the HCl + Static group. ^#P < 0.05, vs. the Stretch + HCl group. (a) The expression of E-cadherin, cytokeratin-8, vimentin and α-SMA in BEAS-2B cells was measured by Western blot analysis. (b and c) The expression levels of E-cadherin, cytokeratin-8, vimentin and α-SMA were confirmed by cell immunostaining. Bar = 75 µm. (d-g) Male C57BL/6 mice were intratracheally instilled with HCl (pH 1.2, 2 ml/kg). Apyrase (4 U/ml) was administered by intratracheal instillation 24 h after HCl instillation. Then, the mice were subjected to MV for 2 h. After 14 days, lung tissues were harvested for subsequent assays. n = 6 each, *P < 0.05, **P < 0.01 vs. the HCl group; ^#P < 0.05, ^##P < 0.01, vs. the HCl + MV group. (d-f) The lung injury score and lung fibrosis score were determined based on lung histological analysis with HE staining and Masson’s trichrome staining. Original magnification, 200 ×. Lung tissue levels of (g) hydroxyproline and (h) collagen 1 on day 14. (i) The protein expression of E-cadherin, cytokeratin-8, vimentin and α-SMA was measured by Western blot analysis. A representative image from three independent experiments is presented. (j and k) The expression of E-cadherin, cytokeratin-8, vimentin and α-SMA was confirmed by immunostaining. Bar = 40 µm.

Fig. 6

Piezo1-mediated ATP release drove mechanical stretch-enhanced pulmonary fibrosis and EMT in vitro and in vivo.(a- c) BEAS-2B^Piezo1-/- cells underwent HCl (pH 4) stimulation for 30 min. After 4 h, the cells were then exposed to mechanical stretch (20 % elongation) for 48 h in the presence of exogenous ATP (100 μM). n = 6 each, *P < 0.05 vs. the BEAS-2B^Piezo1-/- +HCl + stretch group. (a) The expression of E-cadherin, cytokeratin-8, vimentin and α-SMA in BEAS-2B cells was measured by Western blot analysis. (b and c) The expression levels of E-cadherin, cytokeratin-8, vimentin and SMA were confirmed by cell immunostaining. Bar = 75 µm. (d-g) Piezo1^CKO mice were intratracheally instilled with HCl (pH 1.2, 3 ml/kg). Exogenous ATP (100 μM/mouse) was administered by intratracheal instillation 24 h after HCl instillation. Then, the mice were subjected to MV for 2 h. After 14 days, lung tissues were harvested for subsequent assays. n = 6 each, *P < 0.05, **P < 0.01 vs. the Piezo1^CKO + HCl + MV group. (d-f) The lung injury score and lung fibrosis score were determined based on lung histological analysis with HE staining and Masson’s trichrome staining. Original magnification, 200 ×. Lung tissue levels of (g) hydroxyproline and (h) collagen 1 on day 14. (i) The protein expression of E-cadherin, cytokeratin-8, vimentin and α-SMA was measured by Western blot analysis. A representative image from three independent experiments is presented. (j and k) The expression of E-cadherin, cytokeratin-8, vimentin and SMA was confirmed by immunostaining. Bar = 40 µm.