Rat AEC Type II (ATCC, L2 cell line # CCL-149™) were used. L2 cells at passage 7 were seeded onto six-well BioFlex Collagen I coated plates (Flexcell International Corporation, Burlington, NC, United States) at a density of 1.5 × 10⁵ cells/well in Dulbecco’s modified Eagle medium (DMEM, with High Glucose, L-glutamine, Phenol Red and Sodium Pyruvate, Gibco, # 11995073) supplemented with 10% fetal bovine serum (FBS, Gibco) and 50 μg/mL gentamycin sulfate (Gibco, # 15710049). Cells were incubated at 37°C, 21% O2 and 6.5% CO₂ for 24 h. Sixteen hours before stretch experiments, cells were washed twice with sterile phosphate-buffered saline (PBS) and then incubated with DMEM (without Phenol Red, Merck Sigma-Aldrich, #D1145) containing 1% FBS, 50 µg gentamycin sulfate/mL, 4 mM L-alanyl-L-glutamine (Merck Sigma-Aldrich, # G8541), and 1mM Sodium Pyruvate (Gibco, # 11360070).

Verteporfin (Merck Sigma-Aldrich, SML0534) stock solution of 1 M was prepared in dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions. One hour prior to the YAP inhibitor experiment, cells were pretreated with 2 µM verteporfin. An equal volume of DMSO was added to control cells.

We used a custom-made stretching device for cell cyclic-stretching, as previously described (Rentzsch et al., 2017; Ferreira et al., 2022) Briefly, the device utilizes three cylindrical intenders powered by a stepper motor (vertical motion, Maxon Motor AG, Sachseln, Switzerland) to apply a homogeneous cyclic equibiaxial stretch on three wells of a BioFlex plate with silicone membrane, while the other three wells served as non-stretched controls. The driving motor and stretching parameters were controlled by a custom-made program (LabView, National Instruments, Austix, TX, United States). The stretch group was subjected to a magnitude of 30% strain at 0.5 Hz frequency with a stretching/relaxation ratio of 1:1 (sinusoidal pattern) for 1 or 4 h, as previously describted (Ferreira et al., 2022).

Total RNA was extracted with the NucleoSpin RNA Plus, Mini kit (Macherey-Nagel, Düren, Germany, # 740984.50) according to the manufacturer’s protocol. RNA concentrations were evaluated using a spectrophotometer, and the purity was assessed based on the ratio of absorbance at 260 nm and 280 nm (A260–A280). Subsequently, RNA was reversely transcribed using the qScript Ultra SuperMix (Quantabio, Beverly, MA, United States, #95217-100) and the resulting cDNA was used for real-time polymerase chain reaction (PCR) with the PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA, United States, # 95071). The gene expression of IL-6, Cyr61/CCN1, and CTGF/CCN2 was quantified by real-time PCR on the PCR MyiQ™ 2 Cycler (Biorad, Kabelsketal, Germany), using the IQ 5 software (version: 2.1.97.1001) and the following conditions: 95°C for 15 s, followed by 45 cycles at 95°C for 5 s, 60°C for 15 s, and extension at 68°C for 10 s. The primers (Eurofins Genomics, Ebersberg, Germany) are listed in Table 1. Cycle threshold (Ct) values of target genes were normalized to the housekeeping gene β-Actin.

Cells were washed once with ice-cold PBS supplemented with 1 × protease inhibitor cocktail (Roche Applied Science, # 11836153001) and incubated in the hypotonic buffer containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM KCl, 1.5 mM MgCl₂, and 1 mM DL-Dithiothreitol (DTT) on ice for 10 min. Cells were lyzed by passaging 10 times through a 27G needle attached to a1-mL syringe pipetted for 40 s, 14000 x g, 4°C. Supernatants cytosolic protein were collected. The nuclear pellet was resuspended and homogenized in buffer (pH 7.5) containing 50 mM Tris–HCl buffer, 150 mM sodium chloride, 2 mM Ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, supplemented with 1 × protease inhibitor cocktail (Roche Applied Science, # 11836153001) and incubated on ice for 30 min. Subsequently, insoluble proteins were harvested from the nuclear extract by high-speed centrifugation (20 min, 14,000 × g, 4°C).

Cells were homogenized in lysis buffer (pH 7.5) containing 50 mM Tris–HCl, 150 mM sodium chloride, 2 mM Ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, supplemented with 1 × protease inhibitor cocktail (Roche Applied Science, # 11836153001). The denatured proteins (10 μg each well) were separated on a sodium dodecyl sulfate–polyacrylamide gel (4%–20% Mini-PROTEAN TGX Gels, Bio-Rad Laboratories, Hercules, CA, United States, #4561094) and transferred to polyvinylidene fluoride membrane (0.2 µm, Bio-Rad, #1704272) using Semi-Dry blotting system (TransBlot^® Turbo™ Transfer System, Bio-Rad). The membranes were blocked in Tris-buffered saline containing 0.1% Tween 20% and 5% low fat milk, and then incubated with the following primary antibodies overnight at 4°C: anti-IL-6 (1:500, Santa Cruz Biotech, Inc., Dallas, TX, United States, #sc-57315), anti-GAPDH (1:20,000, Calbiochem, San Diego, CA, United States, #CB1001), anti-YAP (1:2,000, Cell Signaling Technology, Danvers, MA, United States, #14074), anti-Phospho-YAP Ser127 (pYAP, 1:2,000, Cell Signaling Technology, #13008), anti-Lamin A/C (1:1,000, Cell signaling, #4777T), anti-α-Tubulin (1:1,000, Thermo Fisher Scientific, Waltham, MA, United States, #A11126), anti-Cyr61/CCN1 (1:1,500, Proteintech, Rosemont, IL, United States, #26689-1-AP), anti-CCN2/CTGF (1:700, Santa Cruz, #Sc14939), anti-Vinculin (1:500, Bio-Rad AbD Serotec, Neuried, Germany, #B1017), andβ-Actin (1:20,000, Merck Sigma-Aldrich,# A3854). After washing, membranes were incubated with secondary antibodies anti-mouse (GE Healthcare, #381334, at a dilution of 1:1,000 for IL-6, Lamin A/C, and α-Tubulin, a dilution of 1:4,000 for GAPDH), or goat anti-rabbit (BioRad, #1721019, at a dilution of 1:1,000 for Cyr61/CCN1, 1:2,000 for YAP and pYAP), or mouse anti-goat (Jackson ImmunoResearch, #205-035-108, at a dilution of 1:1,000 for CTGF/CCN2) at room temperature for 1 h. The blots were developed with WesternBright Chemiluminescence Substrate Quantum (Advansta, San Jose, CA, United States, #541013) and visualized employing a Fujifilm LAS-3000 Luminescent Image Analyzer (Fuji Film, Minato, Japan, 2006). Densitometric analysis of the protein bands was performed using the ImageJ software (Schneider et al., 2012).

To examine the effects of 30% cyclic stretch on cytoskeletal remodeling, stress fibers and intercellular junctions were visualized by immunofluorescence of F-actin (filamentous actin) and Pan-cadherin (all members of the cadherin family of proteins including N-cadherin, E-cadherin, P-cadherin, and R-cadherin). Briefly, cells were cultured in six Flexcell plates (Flexcell International Corporation, Burlington, NC, United States) for 48 h until they reached confluence. Cells were washed twice with ice-cold PBS and fixed with 4% paraformaldehyde (PFA) on ice for 5 min. The PFA was washed away twice with PBS. Subsequent permeabilization was performed by incubating cells with 0.1% Triton X-100 in PBS at room temperature for 5 min. Afterwards, cells were blocked with 3% bovine serum albumin in PBS at room temperature for 1 h, followed by overnight incubation with Invitrogen™ Alexa Fluor™ 488 Phalloidin (1:500, Molecular Probes, Inc., Eugene, OR, United States, # A12379) or primary antibodies against YAP (1:200, Cell Signaling Technology, #14074), anti-Surfactant Protein C (1:200, Merck Millipore Corporation, Burlington, MA, United States, #AB3786), and Pan-cadherin (1:300, Merck Sigma-Aldrich, C1821) at 4°C. After washing with PBS, cells were incubated with secondary antibodies including Alexa Fluor 488 goat anti-mouse IgG (1:1,000, Thermo Fisher Scientific, Waltham, MA, United States, # A-11034) and Alexa Fluor 594 goat anti-mouse IgG (1:200, Thermo Fisher Scientific, # A-11005) at room temperature for 1 h, as well as 4 h, 4’,6-diamidino-2-phenylindole (DAPI, 1:200, Merck Sigma-Aldrich, # D-9542) at room temperature for 30 min. The stained cells attached to the silicone membranes were cut off the plates and mounted inverted on slides using the anti-fade mounting reagent (MOWIOL, Calbiochem, # 475904).

Immunofluorescence images were acquired with a Zeiss LSM880 confocal laser scanning microscope with Airyscan using a ×40 oil immersion objective at the Cellular Imaging Facility of the Medical Theoretical Centre, Faculty of Medicine of the TUD Dresden University of Technology. Fluorescence was detected at the wavelengths: 594 nm (Alexa 594), 488 nm (Alexa 488), and 405 nm (DAPI). Image processing was performed by ImageJ (Schneider et al., 2012).

Data are reported as median ± interquartile range (IQR). Normal distribution of data was tested by the Shapiro-Wilk test. Comparisons among groups were conducted with Kruskal–Wallis test. The data were analyzed using IBM SPSS Statistics (Version 26) (IBM Corp, 2017). Statistical significance was accepted at p < 0.05.

The identity of the type II alveolar epithelial cells was confirmed by immunostaining with the specific marker SP-C (Supplementary Figure S1), as previously described (Ferreira et al., 2022). Application of 30% cyclic stretch for 1 hour and 4 hour caused the remodeling of the cytoskeleton and membrane deformation of AEC II cells. Cyclic stretch (30%) resulted in changes in cell morphology and orientation of the cytoskeleton (rearrangement of actin bundles, paracellular gaps and disrupted intercellular contacts). Representative images of F-actin immunostaining (labelled in green) are shown in Figure 1. In addition, intercellular junctions and cell-cell contacts were impaired in cells exposed to 30% cyclic stretch, compared to non-stretched controls, as shown by pan-cadherin staining (Figure 2).

Protein expression of total (total YAP) and at Ser127 phosphorylated (p-YAP) YAP protein were analyzed by Western blot in total cell lysate (Figure 3). We did not detect a significant upregulation of total YAP or p-YAP after 1 h of 30% cyclic stretch, while both total YAP, p-YAP protein and p-YAP/YAP ratio decreased after 4 h of cyclic stretch compared to non-stretched control.

Next, we analyzed the subcellular localization of YAP protein in response to 1 and 4 h of cyclic stretch was analyzed by immunofluorescence (Figure 4). Cells were labeled with a specific antibody against YAP and analyzed by immunofluorescence. After 1 h the ratio between nuclear and cytosolic YAP increased compared to the control cells.

These findings were further confirmed, quantified by analyzing the YAP protein expression using Western blot in cytosolic and nuclear protein fractions of AEC II exposed to cyclic stretch (Figure 5). After 1 h of 30% cyclic stretch, nuclear YAP was significantly upregulated. We did not detect any statistical difference in nuclear YAP after 4 hours of 30% cyclic stretch. Furthermore, the nuclear/cytosolic YAP ratio was increased after 1 and 4 h compared to control.

Next, we analyzed the mRNA and protein expression of two YAP target genes, Cyr61/CCN1 and CTGF/CCN2. Both genes and proteins were increased by one and 4 h of cyclic stretch except for Cyr61/CCN1 protein in 1 h stretch (Figure 6).

Figure 7 shows the quantitative RT-PCR and Western blot data of cellular IL-6 mRNA and protein expression in response to 30% cyclic stretch. Compared to static control, after 1 and 4 h of 30% cyclic stretch, IL-6 mRNA relative expression was upregulated (Figure 7A). The IL-6 protein expression was significantly induced after 1 h (Figure 7B) compared to the static control.

Finally, we analyzed the impact of YAP inhibition on the expression of its target genes in response to cyclic stretch in alveolar epithelial cells (Figure 8). Cells were pretreated with the YAP inhibitor verteporfin (VP, 2 µM) prior to the exposure to cyclic stretch for 1 h. No changes were detected on the Cyr61/CCN1 and CTGF/CCN2 mRNA expression (Figure 8).

In contrast, Cyr61/CCN1 protein upregulation was significantly blocked by YAP inhibition after 1 h of cyclic stretch (Figure 9). Furthermore, the CTGF/CCN2 protein upregulation was almost reduced to baseline levels (Figure 9). VP treatment without cyclic stretch (as control) did not affect the basal Cyr61/CCN1 and CTGF/CCN2 protein expression.

Basal and stretch-induced IL-6 mRNA expression was not affected by VP pretreatment (Figure 10A). However, the IL-6 protein expression was downregulated by VP under static conditions and after 1 h of 30% cyclic stretch (Figure 10B).

The present study has several limitations. First, in vitro conditions do not fully reproduce the complex environment of the lung parenchyma in vivo, such as the three-dimensional architecture, elasticity, and stiffness, as well as the presence of the surrounding substrate. Second, although our device was designed to apply an equibiaxial strain field, a fraction of cells in the well’s periphery were exposed to strains of less than 30% due to one side fixation at the edge. This introduces a minor uncertainty about the observed mechanical threshold and partial inhomogeneity. Third, although VP is undoubtedly the most popular YAP inhibitor within the scientific community, the molecular details, of how VP binds to YAP, are still poorly understood. As an example, the tumor-inhibitory effects of VP are reported to be YAP-independent (Zhang et al., 2015; Dasari et al., 2017). In these studies, the antiproliferative and cytotoxic effects of VP were YAP-independent in endometrial cancer cells.

In rat alveolar epithelial cells, 30% cyclic stretch activated the expression of YAP target genes Cyr61/CCN1, CTGF/CCN2, and proinflammatory IL-6, which was attenuated by a YAP inhibitor. These findings indicate that YAP might be involved in the inflammatory alveolar epithelial response to ventilator-induced lung injury. Its inhibition might provide a novel therapeutic option for the treatment of ventilator-induced biotrauma.