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. 2022 Dec 13;53:175–186. doi: 10.1016/j.jare.2022.12.006

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

Xiang-Zhi Fang a,b, Min Li c, Ya-Xin Wang a,b, Pei Zhang d, Miao-Miao Sun b, Jia-Xin Xu a,b, Yi-Yi Yang b, Ya-Jun He a,b, Yuan Yu a,b, Rui-Ting Li a,b, Ting Zhou a,b, Le-Hao Reng a,b, De-Yi Sun a,b, Hua-Qing Shu a,b, Shi-Ying Yuan a,b, Ji-Qian Xu a,b,⁎, You Shang a,b,⁎
PMCID: PMC10658225  PMID: 36526145

Graphical abstract

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Keywords: Mechanical ventilation, Piezo1, Pulmonary fibrosis, Acute respiratory distress syndrome

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-hit model of MV after acid aspiration-induced lung injury in 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. Pharmacological inhibition or knockout 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 novel strategy for the treatment of MV-exacerbated ARDS-associated pulmonary fibrosis.

Introduction

Acute respiratory distress syndrome (ARDS) is a fatal progressive disease involving hypoxemic respiratory failure that is characterized by severe pulmonary inflammation, hypoxemia, and pulmonary edema in critically ill patients [1]. ARDS can be directly induced through a direct insult to the parenchyma (pneumonia, aspiration, contusion) or indirectly through a systemic inflammatory response (sepsis, trauma, bypass surgery). Pulmonary fibrosis is a major cause of the poor prognosis of ARDS [2]. The main pathological change that occurs during the early stage of ARDS (<7 d) is conventionally believed to be exudation due to lung injury, whereas the abundance of collagen fibers starts to increase 7 days later, and pulmonary fibrosis develops gradually [3], [4]. Subsequent studies [5], [6], however, have revealed that pulmonary fibroproliferation is initiated early in ARDS and is related to clinical outcome.

While mechanical ventilation (MV) is an essential life-saving treatment for patients with ARDS, it has a strong potential to cause lung epithelial cell damage and exacerbate pre-existing lung injury in is a process known as ventilator-induced lung injury (VILI) [7], [8]. Additionally, prolonged MV can lead to disorganized remodeling of lung epithelial cells, which exacerbates ARDS-associated lung fibrosis and contributes to poor quality of life in long-term survivors of ARDS [9], [10], [11].

Epithelial-mesenchymal transition (EMT), a biological process during which epithelial cells lose their epithelial markers and acquire mesenchymal characteristics, is essential in epithelial repair after injury [12]. Increasing evidence suggests that EMT is the basis of pulmonary fibrosis [13], [14]. Interestingly, Heise et al. found that mechanical stretch resulted in EMT in lung epithelial cells [15]. Furthermore, several recent studies in a two-hit model of ARDS induced by MV after HCl aspiration in mice have suggested that impaired mechanical stretch is associated with the development of EMT and thus is essential for MV-associated pulmonary fibrosis [16], [17], [18]. Although EMT plays a critical role in MV-associated pulmonary fibrosis, the underlying molecular mechanisms that mediate the EMT process in response to mechanical stretch stimulation are not well understood.

Piezo proteins (Piezo1 and Piezo2) are relatively recently identified mechanosensitive channels activated by mechanical force [19]. Piezo proteins translate extracellular mechanical forces to intracellular molecular signaling cascades through a process known as mechanotransduction [20]. In particular, Piezo1 is highly expressed in tissues subjected to regular deformation or shear force, such as the lungs, bladder, skin and vasculature endothelium [21]. However, the role of Piezo1 in MV-exacerbated EMT and pulmonary fibrosis is unclear. It has been reported that Piezo1 is essential for regulating bladder distention, red blood cell volume, and vascular development and that its protective mechanism may be due to its role in mediating Ca2+ influx [21]. Given that Piezo1 is involved in the deleterious effects of mechanical force [22], [23], we hypothesized that activation of the ion channel Piezo1 plays an important role in the development of EMT and pulmonary fibrosis in response to mechanical stretch in ARDS.

To test this hypothesis, we used a two-hit model of lung fibrosis that involves cyclic mechanical stretch following HCl exposure. It has been suggested that “two-hit” model is closer to the real clinical situation. HCl is used to reproduce ARDS caused by gastric acid aspiration.

Materials and methods

Cell culture

Human lung epithelial cells (BEAS-2B; American Type Culture Collection, USA) were cultured in BEGM with SingleQuot kit additives (product # CC-3170, Lonza, Walkersville, MD) at 37 °C in a humidified atmosphere containing 5 % CO2. The cell lines included in this study were authenticated by STR profiling and tested for mycoplasma contamination.

Mechanical stretch system and in vitro cell studies

Human lung epithelial cells (BEAS-2B) (4.0 × 105/well) were grown on untreated six-well plates with a flexible bottom (BioFlex culture plates) until they reached 85 % cell density. Then, the cells were exposed to BEGM (pH 4.0). After 30 min of stimulation, the cells were rinsed twice with PBS and cultured in BEGM with SingleQuot kit additives for 4 h. Afterward, the Flexercell Tension Plus system (FX-5000 T; Flexcell International, USA) was used to apply mechanical stretch to the BEAS-2B cells (20 % amplitude, 30 cycles/min, 48 h) (Fig. 1a). In some experiments, the Piezo1 antagonist GsMTx4 (2.5 µM) (ab141871; Abcam, USA) or the ATP-hydrolyzing enzyme apyrase (A6535; Sigma, USA) was administered 30 min prior to mechanical stretch. In another experiment, cells were stimulated with ATP (100 μM) (HY-108666, MCE, USA) or Yoda1 (25 µM) (SML1558; Sigma, USA).

Fig. 1.

Fig. 1

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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.

Ethics statement

All experiments involving animals were conducted according to the ethical policies and procedures approved by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology.

Generation of conditional Piezo1 knockout mice

To generate male mice with tamoxifen-inducible Piezo1 deletion specifically in the lung epithelial cells (Piezo1CKO), Piezo1F/F mice (Piezo1tm2.1Apat/J; Jackson Laboratory) were crossed with Sftpc-CreERT mice (Jackson Laboratory). Genotyping was undertaken by PCR using primers specific for Cre and Piezo1 (STable 2). To knock out Piezo1, 3-week-old Piezo1F/F; Sftpc-CreERT mice were given a tamoxifen suspension (0.1 ml of 10 mg/ml) (HY-Y1888, MCE, USA) by intraperitoneal injection for 7 d. Age- and weight-matched Piezo1F/F mice were used as controls for Piezo1SFTPC-CRE knockout mice. Successful Piezo1 knockout in lung epithelial cells was confirmed by genotyping and immunofluorescent double labeling.

Two-hit model of MV after acid aspiration-induced lung injury in mice

All mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). After orotracheal intubation, HCl (pH 1.2) was instilled intratracheally (2 ml/kg). Mice in the control group were intratracheally injected with saline. After 48 h, the mice were then orotracheally intubated and mechanically ventilated for 2 h (ventilator model: Harvard, 55–7062), and the ventilation parameters were set as described in our previous study [24]. The mice were monitored for survival for 14 days after MV began (Fig. 1e).

In some experiments, the Piezo1 antagonist GsMTx4 (270 µg/kg, intraperitoneally) or the ATP-hydrolyzing enzyme apyrase (4 U/ml, intratracheally) was administered 30 min prior to MV. In another experiment, ATP (100 µM/mouse, intratracheally) was administered intratracheally. In an additional experiment, recombinant adeno-associated virus (rAAV) was administered by tracheal instillation to deliver shRNA targeting Piezo1, which reduced Piezo1 expression in the lung tissue.

Statistical analysis

The data are presented as the mean ± SED. Comparisons between two groups were performed using independent sample t tests. One-way ANOVA and the Bonferroni post hoc test were used to compare more than two groups. All statistical analyses were performed using Prism 5 (GraphPad Software, USA), and a P value<0.05 indicated statistical significance.

Results

MV exacerbated pulmonary fibrosis and EMT

We first examined whether mechanical stretch would enhance EMT in vitro by investigating changes in EMT markers. The stimulation of BEAS-2B cells with HCl or mechanical stretch caused a decrease in E-cadherin and cytokeratin-8 expression and an increase in alpha-smooth muscle actin (α-SMA) and vimentin expression. These changes were enhanced when the cells were stimulated with HCl before mechanical stretch (Fig. 1b). To further confirm the EMT profile, immunofluorescent double labeling was performed. The expression of cytokeratin-8 and E-cadherin decreased, whereas α-SMA and vimentin expression increased after mechanical stretch or HCl stimulation (Fig. 1c and d). These effects were more pronounced when the cells were exposed to HCl after mechanical stretch.

To investigate the effects of MV on ARDS-associated fibrosis, a two-hit model of MV after acid aspiration-induced lung injury in mice was established. On day 14, mice in both the HCl group and MV group demonstrated thickening of the alveolar septa and the increased infiltration of mononuclear cells (Fig. 1f). As an important indicator of pulmonary fibrosis, fibrillar collagen deposition was evaluated using Masson’s trichrome staining. Masson’s staining showed a marked increase in the deposition of blue-stained collagen in the lung tissues of mice in both the HCl group and MV group (Fig. 1f). Furthermore, lung injury and fibrillar collagen deposition were more apparent in the HCl + MV group than in the HCl group (Fig. 1g and h). Additionally, the levels of hydroxyproline and collagen I were measured with a commercial kit. The results showed that HCl or MV alone enhanced hydroxyproline and collagen 1 levels. Moreover, the upregulation of collagen I and hydroxyproline in the HCl + MV group was more pronounced than that in the other groups (Fig. 1i and j). Western blotting and immunofluorescence staining were performed to examine alterations in the levels of EMT markers in lung tissue. As shown in Fig. 1k, HCl or MV decreased the protein levels of E-cadherin and cytokeratin-8 to varying degrees and increased the protein levels of α-SMA and vimentin to varying degrees. Furthermore, MV dramatically enhanced the changes in EMT marker levels induced by HCl. The results of the immunofluorescence assay were generally consistent with the Western blotting results. HCl or MV promoted EMT to different degrees, and MV dramatically enhanced the HCl-induced increase in EMT (Fig. 1l and m). Taken together, these results indicated that MV exacerbated pulmonary fibrosis and EMT.

Mechanical stretch induced Piezo1 activation in human lung epithelial cells

Given its importance in mechanotransduction, we sought to investigate whether Piezo1 mediates MV-induced pulmonary fibrosis. We first investigated the cellular localization of Piezo1 in BEAS-2B cells and lung tissue by immunofluorescence staining. We found that Piezo1 was expressed in the plasma membrane and cytoplasm near the nucleus in BEAS-2B cells (Fig. 2a). Confocal microscopy was used to visualize frozen sections of murine lung tissue, and the results showed that Piezo1 was expressed in all epithelial cells in the lung tissue, including bronchial epithelial cells (Fig. 2a). Additionally, we analyzed the expression of known mammalian mechanosensory ion channels [25], [26], [27], [28], [29], including Piezo family members, KCNK4, TRPA1, stoml3, TRPC3, TRPC6, and TRPV4, in unstimulated BEAS-2B cells by RT-PCR. Piezo1 was the most abundantly expressed of the tested genes in unstimulated BEAS-2B cells (Fig. 2b). These results showed that Piezo1 was indeed present in unstimulated bronchial airway epithelial cells.

Fig. 2.

Fig. 2

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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-2BWT cells and BEAS-2BPiezo1-/- 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.

We next examined changes in the expression of Piezo1 in bronchial airway epithelial cells exposed to mechanical stretch. We found that mechanical stretch significantly upregulated Piezo1 expression in HCl-stimulated BEAS-2B cells (Fig. 2c). Then, we examined functional changes in Piezo1 through Ca2+ assays. Our results showed that mechanical stretch increased intracellular Ca2+ levels in BEAS-2B cells loaded with the calcium indicator Fluo-3AM (Fig. 2d). To further define the functional changes in Piezo1 in BEAS-2B cells, we generated a Piezo 1 knockout BEAS-2B cell line (BEAS-2BPiezo1-/-) by CRISPR/Cas9-based gene editing. Successful deletion of Piezo1 expression was verified by Sanger sequencing and Western blot analysis of Piezo1 (Figure S1 a and b). We found that the increase in intracellular Ca2+ levels in BEAS-2B cells was strongly reduced after pretreatment with GsMTx4 or Piezo1 knockout (Fig. 2d). To determine which pool contributed to the elevated intracellular Ca2+ levels, we used an experimental strategy that is commonly used in Ca2+ signaling research: incubation in Ca2+-free medium to block extracellular Ca2+ entry. We found that incubation in Ca2+-free medium inhibited the mechanical stretch-induced increase in intracellular Ca2+ levels (Fig. 2d). Additionally, we measured real-time Ca2+ imaging in BEAS-2B cells incubated with Yoda1 and similar results were obtained. (Fig. 2e-g). This result indicated that mechanical stretch indeed induced Piezo1 activation in bronchial airway epithelial cells.

Genetic deletion of Piezo1 protected against MV-exacerbated ARDS-associated pulmonary fibrosis and EMT in vitro and in vivo.

Next, we sought to uncover whether Piezo1 was required for mechanical stretch-enhanced EMT. To assess the function of Piezo1, we exposed BEAS-2BWT cells and BEAS-2BPiezo1-/- cells to HCl and mechanical stretch stimulation for 48 h. Relative to BEAS-2B cells, BEAS-2BPiezo1-/- cells displayed a drastic reduction in α-SMA and vimentin levels in response to mechanical stretch for 48 h. Moreover, the expression of cytokeratin-8 and E-cadherin was also increased in BEAS-2BPiezo1-/- cells (Fig. 3a-c). A similar effect was observed in BEAS-2B cells treated with GsMTx4 (Figure S2 a-c). Taken together, our data demonstrated that Piezo1 was critical for mechanical stretch-induced EMT.

Fig. 3.

Fig. 3

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Piezo1 knockdown inhibited MV-enhanced lung fibrosis and EMT in vitro and in vivo.(a -c) BEAS-2BWT cells and BEAS-2BPiezo1-/- 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-2BWT + 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) Piezo1CKO mice and Piezo1F/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 Piezo1F/F + HCl group; #P < 0.05, ##P < 0.01 vs. the Piezo1F/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.

To probe the function of the Piezo1 protein in MV-exacerbated pulmonary fibrosis in vivo, we generated mice in which Piezo1 was conditionally knocked out in the lung epithelial cells (Piezo1CKO mice). The effect of Piezo1 knockout was examined through genotyping and immunofluorescence staining (Figure S1 c and d). As shown in Fig. S1d, the expression of Piezo1 protein was noticeably reduced on the lung bronchial airway epithelial cells of Piezo1CKO mice, which indicated deletion of the Piezo1 in lung epithelial cells. Piezo1 knockout in lung epithelial cells significantly attenuated lung injury and collagen deposition in the lung tissue, as detected by hematoxylin and eosin (H&E) and Masson’s trichrome staining (Fig. 3d-f). The levels of hydroxyproline and collagen 1 were also downregulated in Piezo1CKO mice (Fig. 3 g and h). Furthermore, we then performed Western blotting and immunofluorescence staining to detect EMT markers. Consistently, the mechanical stretch-induced loss of cytokeratin-8 and E-cadherin expression in the lung tissues was largely prevented, whereas the expression of α-SMA and vimentin was dramatically decreased in Piezo1CKO mice (Fig. 2i-k). GsMTx4 treatment or Piezo1 knockdown using rAAV in mice had an effect similar to that observed in the Piezo1CKO mice (Figure S2, Figure S3). Taken together, our data demonstrated that Piezo1 was critical for MV-enhanced lung fibrosis and EMT.

Mechanical stretch-induced ATP release in human lung epithelial cells depended on Piezo1

We next determined how Piezo1 regulates EMT and pulmonary fibrosis. The release of extracellular ATP (eATP) from damaged cells is essential for lung inflammation and bleomycin-induced pulmonary fibrosis and has been recently implicated as an important downstream signaling event in Piezo1 activation [30], [31], [32]. Thus, we wanted to explore whether Piezo1 mediates mechanical stretch-enhanced EMT by promoting ATP release. First, we found that the Piezo1 agonist Yoda1 rapidly induced ATP release from BEAS-2B cells. In addition, GsMTx4, a Piezo1 antagonist, strongly inhibited Yoda1-induced ATP release (Fig. 4a). Furthermore, we also found that mechanical stretch could induce ATP release from BEAS-2B cells, and this effect was strongly reduced after pretreatment with GsMTx4 or Piezo1 knockout in the BEAS-2B cells (Fig. 4b). To exclude the contribution of cellular damage to the increase in ATP release observed under mechanical stretching, we tested whether mechanical stretch affected lactate hydrogenase (LDH) activity in the supernatant. As shown in Fig. 4c, no significant change in LDH activity was observed. In vivo, the amount of ATP released in the bronchoalveolar lavage fluid (BALF) caused by MV gradually increased and peaked at 6 h, and MV-induced ATP release into the BALF was significantly inhibited by GsMTx4 treatment or Piezo1 knockout (Fig. 4d and 4e). These results indicated that the mechanical stretch-induced increase in ATP release in the BEAS-2B cells depended on Piezo1.

Fig. 4.

Fig. 4

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Mechanical stretch-induced ATP release from BEAS-2B cells depended on Piezo1.(a-c) BEAS-2BWT cells and BEAS-2BPiezo1-/- cells were exposed to 25 μM Yoda1 or (b) mechanical stretch (20 % elongation) after HCl (pH 4) stimulation for 30 min. BEAS-2BWT 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-2BWT 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 Piezo1CKO 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. Piezo1F/F. (f and g) BEAS-2BWT cells and BEAS-2BPiezo1-/- 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.

Next, we depleted and blocked intracellular Ca2+ by loading BEAS-2B cells with the high-affinity Ca2+ chelator BAPTA-AM and then subjected the cells to mechanical stretch. Preventing the increase in intracellular Ca2+ levels due to mechanical stretch stimulation decreased eATP release from BEAS-2B cells. In contrast, BEAS-2BPiezo1-/- cells did not exhibit inhibited eATP release after pretreatment with BAPTA-AM. Overall, in comparison to BEAS-2BWT cells, BEAS-2BPiezo1-/- cells produced lower levels of eATP (Fig. 4f). To further understand the role of Piezo1-dependent Ca2+ influx in eATP release, we used EGTA to block extracellular Ca2+ entry. After the cells were incubated in Ca2+-free medium, the level of eATP was reduced (Fig. 4g). These findings suggested that Yoda1 or mechanical stretch contributed to ATP release via Piezo1-dependent Ca2+ influx.

Exogenous ATP promoted pulmonary fibrosis and EMT in vitro and in vivo

Then, we investigated whether exogenous ATP could also promote ARDS-associated pulmonary fibrosis and EMT. To this end, cells were treated with the nonhydrolyzable ATP analog ATPγS (γ-ATP) and/or HCl. Interestingly, a single application of γ-ATP did not significantly affect the expression of EMT markers (Fig. S4a-c). However, exogenous γ-ATP treatment significantly enhanced the HCl-induced changes in EMT marker levels in vitro (Fig. S4a-c). Similarly, a single instillation of exogenous γ-ATP did not cause pulmonary fibrosis in vivo (Fig. S4d-f). However, exogenous γ-ATP significantly exacerbated lung injury and collagen deposition in lung tissue exposed to HCl (Fig. S4d-f). Compared to those in the HCl group, the levels of hydroxyproline and collagen 1were also upregulated in mice treated with HCl and exogenous ATP (Fig. S4g and h). Furthermore, exogenous γ-ATP enhanced the HCl-induced loss of cytokeratin-8 and E-cadherin and promoted the expression of α-SMA and vimentin in lung tissue (Fig. S4i-k). Taken together, our data demonstrated that exogenous γ-ATP promoted HCl-induced pulmonary fibrosis and EMT in vitro and in vivo.

Exogenous ATP depletion suppressed mechanical stretch-enhanced pulmonary fibrosis and EMT in vitro and in vivo

We next neutralized the increase in exogenous ATP by administering the ATP-hydrolyzing enzyme apyrase to further investigate the function of exogenous ATP in EMT and pulmonary fibrosis (Fig. S5a). We found that eATP depletion from the culture medium prevented the mechanical stretch-enhanced loss of cytokeratin-8 and E-cadherin and inhibited the mechanical stretch-enhanced expression of α-SMA and vimentin in BEAS-2B cells (Fig. 5a-c). Additionally, we used tracheal instillation of apyrase to reduce the concentration of ATP in the BALF (Fig. S5b). We found that eATP depletion suppressed lung injury and that the abundance of collagen fibers was enhanced by MV (Fig. 5d-f). The mice that were administered apyrase showed significantly decreased concentrations of hydroxyproline and collagen 1 in the lung tissue (Fig. 5g and h). Moreover, eATP depletion abrogated the loss of cytokeratin-8 and E-cadherin and inhibited the expression of α-SMA and vimentin (Fig. 5i-k). Taken together, our data demonstrated that eATP depletion suppressed mechanical stretch-enhanced pulmonary fibrosis and EMT in vitro and in vivo.

Fig. 5.

Fig. 5

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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.

Piezo1-mediated ATP release drove mechanical stretch-enhanced pulmonary fibrosis and EMT in vitro and in vivo

We confirmed that genetic knockdown of Piezo1 reduced mechanical stretch-induced ATP release in vitro and in vivo. Additionally, we found that exogenous ATP depletion suppressed mechanical stretch-enhanced pulmonary fibrosis and EMT in vitro and in vivo. Therefore, we next determined whether exogenous ATP supplementation could compensate for the inhibitory effect of Piezo1 deficiency on mechanical stretch-enhanced pulmonary fibrosis and EMT. We found that exogenous γ-ATP treatment markedly promoted the changes in EMT marker levels induced by mechanical stretch in BEAS-2BPiezo1-/- cells (Fig. 6 a-c). Furthermore, instillation of exogenous γ-ATP significantly exacerbated lung injury and lung collagen deposition in Piezo1CKO mice (Fig. 6d-f). Similarly, instillation of exogenous γ-ATP resulted in a marked increase in the levels of hydroxyproline and collagen 1 compared to those in Piezo1CKO mice treated with HCl and subjected to MV (Fig. 6 g and h). Moreover, exogenous γ-ATP enhanced the loss of cytokeratin-8 and E-cadherin and promoted the expression of α-SMA and vimentin in the lung tissue of Piezo1CKO mice exposed to HCl and subjected to MV (Fig. 6i-k). Taken together, our data demonstrated that Piezo1-mediated ATP release drove mechanical stretch-enhanced pulmonary fibrosis and EMT in vitro and in vivo.

Fig. 6.

Fig. 6

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Piezo1-mediated ATP release drove mechanical stretch-enhanced pulmonary fibrosis and EMT in vitro and in vivo.(a- c) BEAS-2BPiezo1-/- 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-2BPiezo1-/- +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) Piezo1CKO 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 Piezo1CKO + 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.

Discussion

In the present study, we show that mechanical stretch resulting from MV exacerbates ARDS-associated pulmonary fibrosis. Our findings also strongly suggest that Piezo1 plays a critical role in MV-exacerbated pulmonary fibrosis and EMT by mediating Ca2+ influx and ATP release.

Lung epithelial cells are the primary targets of MV and ARDS. EMT is increasingly recognized as part of the process that follows epithelial damage, whereby epithelial cells give rise to myofibroblasts and contribute to the pathogenesis of fibrosis [33]. Notably, mechanical stretch generated during MV initiates EMT, which exacerbates ARDS-associated pulmonary fibrosis and even directly leads to pulmonary fibrosis [15], [34], [35], [36], [37], [38]. Consistently, we also found that both HCl and mechanical stretch caused mild pulmonary fibrosis in vitro and in vivo, were accompanied by mild EMT. Worsened pulmonary fibrosis and EMT were observed in mice exposed to the combination of MV and acid instillation. These results suggested that MV exacerbated ARDS-associated pulmonary fibrosis and EMT.

Piezo1 is a mechanosensitive ion channel that has gained recognition for its role in regulating diverse physiological processes [39]. However, the expression of Piezo1 in pulmonary epithelial cells, which are an important source of myofibroblasts, has not been characterized. In this study, we found that Piezo1 protein expression in unstimulated pulmonary epithelial cells was high. Additionally, mechanical stretch significantly upregulate Piezo1 expression, which suggests that Piezo1 may play a role in mechanical ventilation-exacerbated ARDS fibrosis. Moreover, genetic deletion or pharmacological blockade of Piezo1 prevented the increase in EMT and fibrosis enhanced by MV or mechanical stretch, demonstrating the role of Piezo1 as a key regulator of MV-associated pulmonary fibrosis and EMT.

eATP levels are distinctly low under physiological conditions but can increase several hundred-fold when cells are exposed to injury, stress, or even necrosis [40]. Elevated levels of eATP function as a damage-associated molecular pattern (DAMP) to initiate inflammatory cascades through purinergic receptors, mainly P2XR [41]. Furthermore, eATP is a major endogenous danger signal involved in the establishment of lung inflammation, which leads to fibrosis [30], [42]. Much evidence has shown that ATP release is an important early downstream event in Piezo1 activation [32], [43]. In this study, our findings suggest that Yoda1 or mechanical stretch contributes to ATP release via Piezo1-dependent Ca2+ influx. Previous studies have shown that pannexin channels, which are well known to release ATP, are activated by a wide variety of stimuli, exert diverse effects and can stimulate the influx of extracellular Ca2+ [44], [45], [46]. Thus, we hypothesize that pannexin channels are crucial for Piezo1-dependent ATP release in bronchial epithelial cells exposed to mechanical stretch. However, verifying this hypothesis will require further studies.

Notably, we observed that exogenously administered ATP did not directly induce pulmonary fibrosis or EMT but promoted the increase in pulmonary fibrosis and EMT caused by HCl. Then, we demonstrated that ATP is involved in MV-associated pulmonary fibrosis because enzymatic ATP degradation by apyrase inhibited EMT and collagen deposition. These results present compelling evidence that Piezo1 mediates ATP release and is required for MV-exacerbated ARDS-associated pulmonary fibrosis. The mechanism by which ATP promotes pulmonary fibrosis and EMT is still unclear.

Conclusions

The findings of this study suggest that Piezo1 is crucial in the pathogenesis of MV-exacerbated ARDS-associated pulmonary fibrosis. Its mechanism of action involves the release of intracellular ATP.

Availability of data and materials

The data supporting the conclusions of this article are available from the corresponding author upon request.

Funding

This work was funded by the National Natural Science Foundation of China (No. 81772047, 81971818, 82002026, 82272217 and 82172171).

CRediT authorship contribution statement

Xiang-Zhi Fang: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. Min Li: Formal analysis, Investigation, Project administration. Pei Zhang: Formal analysis, Investigation, Project administration. Miao-Miao Sun: Formal analysis, Investigation, Project administration. Jia-Xin Xu: Formal analysis, Investigation, Project administration. Ya-Jun He: Formal analysis, Investigation, Project administration. Ya-Xin Wang: Conceptualization, Supervision, Funding acquisition, Writing – review & editing. Yuan Yu: Investigation, Data curation. Rui-Ting Li: Investigation, Data curation. Ting Zhou: Investigation, Data curation. Le-Hao Reng: Conceptualization, Methodology, Resources, SupervisionDe-Yi Sun: Conceptualization, Methodology, Resources, SupervisionHua Qing Shu : Conceptualization, Methodology, Resources, SupervisionShi-Ying Yuan: Conceptualization, Methodology, Resources, Supervision.Ji-Qian Xu: Conceptualization, Methodology, Project administration, Supervision, Funding acquisition，Writing – review & editing. You Shang: Conceptualization, Methodology, Project administration, Supervision, Funding acquisition，Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Peer review under responsibility of Cairo University.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jare.2022.12.006.

Contributor Information

Ji-Qian Xu, Email: xjq5456.good@163.com.

You Shang, Email: you_shanghust@163.com.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (1.9MB, docx)

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Associated Data

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Supplementary Materials

Supplementary data 1
mmc1.docx (1.9MB, docx)

Data Availability Statement

The data supporting the conclusions of this article are available from the corresponding author upon request.

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