Reagents

Cycloheximide (CHX, S7418) and MG132 (S2619) were purchased from Selleck (Houston, TX, USA). 3-methyladenine (3-MA, M9281), ammonium chloride (NH4Cl, A9434), chloroquine (CQ, C6628) and the RhoA pathway agonist, U-46619 (D8174) were purchased from Sigma-Aldrich (Munich, Germany).

Construction of Lentiviral Vectors

For in vivo studies, mouse FLRT3 (GenBank accession number NM_001172160) full-length coding sequences were synthesized and subcloned into a GV492 vector (GeneChem). For in vitro studies with HPMECs, human FLRT3 (GenBank accession number NM_013281) full-length coding sequences were used. The results of mouse GV492-FLRT3 and human GV492-FLRT3 vectors were sequenced to confirm correct insertion.

For knockdown experiments, shRNAs targeting FLRT3 or RND3 and a scrambled negative control shRNA were synthesized and cloned into the GV493 vector (GeneChem). The shRNA sequences are given in Table 1. The vectors were co-transfected into 293 T cells using Lipo3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After 72 h, the lentiviruses were harvested and stored at − 80 °C.

Table 1 The shRNA sequences used in this studyAnimal Model and Experimental Design

Sixty-five male C57BL/6J mice (6–8 weeks old, weighing 20–26 g) from Shanghai SLAC Laboratory Animal Co., Ltd. were housed under standard conditions (22 ± 2 °C, 12 h light/dark cycles). All animal procedures were approved by Shanghai Tenth People’s Hospital, Tongji University School of Medicine. To establish the lung I/R model, anesthetized mice (sodium pentobarbital, 50 mg/kg, i.p.) underwent tracheotomy followed by mechanical ventilation (tidal volume 0.6 ml, 120 breaths/min, 1:2 inspiration-to-expiration ratio, 100% oxygen). Left lung ischemia was induced by clamping the left pulmonary hilum for 60 min, followed by reperfusion. Three experimental series were conducted: (1) a time-course study (n = 20) with varying reperfusion durations (0, 60, 120, and 240 min, n = 5 per group); (2) FLRT3 overexpression study (n = 20) comparing Sham, I/R (60 min ischemia, 120 min reperfusion), I/R+LV-control, and I/R+LV-FLRT3 groups (n = 5 per group); and (3) RND3 mechanism study (n = 25) including Sham, I/R+LV-control, I/R+LV-FLRT3, I/R+LV-FLRT3+sh-control, and I/R+LV-FLRT3+sh-RND3 groups (n = 5 per group). For lentiviral interventions, mice received tail vein injections of respective vectors (1 × 109 TU/ml, 100 μl) 3 days before I/R surgery, with successful delivery confirmed by western blot analysis of target protein expression. Sham controls underwent identical procedures without ischemia, while I/R groups experienced 60 min ischemia followed by 120 min reperfusion. The successful delivery was confirmed by examining FLRT3 or RND3 protein expression in lung tissue using western blot. Sham controls underwent identical procedures without ischemia, while I/R groups experienced 60 min ischemia followed by 120 min reperfusion.

Lung Wet-to-Dry Ratio

Pulmonary edema was measured using the lung wet-to-dry ratio method. Briefly, the lower lobe of the left lung was removed and the weight was recorded (wet weight). Samples were dried in a 60 °C incubator for 96 h. Then, the dry weight was measured, and the ratio was calculated.

Immunohistochemistry

Lung tissue sections (5 μm) were deparaffinized, incubated with 3% H2O2 in methanol for 15 min, then boiled in citrate buffer (pH 6.0). Samples were blocked with 5% BSA for 30 min at room temperature and incubated overnight with anti-cleaved caspase-3 antibody (1:400; #9661, Cell Signaling Technology, Danvers, MA, USA). After washing in PBS, samples were incubated with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG at 37 °C for 30 min, then stained using the DAB detection system kit (ZSGB‐Bio, Beijing, China). Cleaved caspase-3 staining was visualized under light microscopy (Olympus, Tokyo, Japan).

Haematoxylin & Eosin (H&E)

Lungs were inflated and fixed (10% neutral-buffered formalin at room temperature for 12–24 h), dehydrated with ethanol and embedded in paraffin. Sections (5 μm) were cut and stained with H&E. The lung injury score was determined based on the following three criteria: (i) aggregation or infiltration of inflammatory cells in vessel walls or air spaces [1 = only wall, 2 = rare cells in air space, 3 = intermediate, and 4 = severe (air space congested)], (ii) hyaline membrane formation and interstitial congestion in the lung [1 = normal lung, 2 = moderate (> 25% of lung section), 3 = intermediate (25%–50% of lung section), and 4 = severe (> 50% of lung section)], and (iii) hemorrhage [0 = absent, and 1 = present] [35]. The sum of the individual scores for each criterion was calculated to obtain the lung injury score for each animal. Sections were independently assessed by two pathologists blinded to the animal grouping.

Evans Blue Dye Assay

For the Evans blue dye assay, 4 ml/kg of 2% Evans blue dye (E2129, Merck KGaA, Darmstadt, Germany) in normal saline was administered through the tail vein 10 h before the animals were humanely killed. After adequate perfusion with normal saline, the Evans blue dye was extracted from the lung using formamide for 18 h at 60 °C. The absorbance of the supernatant was measured at 620 nm on a microplate reader (BioTek Instruments, Winooski, VT, USA) and was reported as the amount of Evans blue dye per wet tissue weight (µg/g).

Transmission Electron Microscopy (TEM)

Lung tissue samples (1–2 mm cubes) were fixed in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde for 2 h at room temperature. Samples were then washed in 0.1 M cacodylate buffer, fixed with 1% OsO4/1.5% KFeCN6 for 1 h, washed in water, and incubated in 1% aqueous uranyl acetate for 1 h. Next, samples were washed in water, dehydrated in increasing concentrations of alcohol, and incubated in propylene oxide for 1 h. Following overnight incubation in a 1:1 mixture of propylene oxide and TAAB Epon, samples were embedded in TAAB Epon and polymerized at 60 °C for 48 h. Sections (60 nm thin) were cut, placed on copper grids, stained with lead citrate and examined under transmission electron microscope (Tecnai-G220-TWIN TEM, FEI, Holland).

Cell Culture

Human pulmonary microvascular endothelial cells (HPMECs), purchased from ATCC (Manassas, VA, USA), were cultured in endothelial cell medium (ECM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco), 1% endothelial cell growth factor (Beyotime Biotechnology, Jiangsu, China), and 100 IU/ml penicillin and streptomycin (Sigma-Aldrich) at 37 °C in a humidified atmosphere of 5% CO2.

To mimic lung I/R in vitro, HPMECs were subjected to hypoxia and reoxygenation (H/R). Briefly, when the cell confluency reached approximately 80%, HPMECs were subjected to 12 h hypoxia (1% O2, 5% CO2 and 94% N2) followed by 4 h reoxygenation (5% CO2, 95% room air) at 37 °C.

Cell Transduction

HPMECs at 70–80% confluence were infected with lentiviral vectors at MOI of 10 in the presence of 5 μg/ml polybrene. After 12 h, the medium was replaced with fresh complete medium. The infection efficiency was confirmed 72 h post-transduction by western blot analysis of target protein expression.

Evans Blue-Albumin Monolayer Permeability Assay In Vitro

HPMECs (1 × 104 cells/well) were seeded into Transwell inserts (pore size 3.0 μm, #3472, Costar, MA, USA) and subjected to various treatments. Permeability was assessed by removing the culture medium from the upper chamber, and replacing it with Evans blue-conjugated albumin (final concentration: 0.67 mg/ml). 4% BSA was added to the lower chamber. The levels of Evans blue‐conjugated albumin in the upper chamber and 4% BSA in the lower chamber were kept at the same height to eliminate the impact of hydrostatic pressure gradient. After incubation for 1 h at 37 °C in 5% CO2, the mixture in the lower chamber was collected, and its absorbance was measured using a microplate reader at a wavelength of 620 nm. The leakage of Evans blue‐conjugated albumin from HPMECs was calculated using a standard curve.

Trans-endothelial Electric Resistance (TEER) Measurements

Endothelial barrier integrity was determined using an electrical resistance system (Millicell‐ERS, MERS00002; Merck Millipore, Germany). The electric resistance of monolayer HMPECs was measured every 4 h, and the TEER was calculated as described by the manufacturer. A high TEER was representative of a high barrier integrity.

Cell Viability

Cell viability was assessed using Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan). Cells were seeded into 96-well plates (1 × 104 cells/ml), and subjected to H/R and lentiviral treatments. Samples were incubated with CCK-8 solution for 2 h at 37 °C, and the absorbance was read using a microplate reader at 450 nm.

Wound Healing Assay

HMPECs were seeded into 6‐well plates at a density of 1.0 × 105 cells/well. Following treatment of the cells, a 1 ml pipette tip was used to scrap through the cellular monolayer. Cells were then washed gently with PBS to remove cell debris. The medium was replaced and cells were incubated for 24 h at 37 °C in 5% CO2. Images were captured by microscopy at the 0 and 24 h time points at the same position for each wound. The migration ability was determined by measuring the width of the scratch wound at the different time points.

Transwell Migration Assay

HPMECs (2 × 104 cells/well) were seeded into Transwell plates (pore size: 8.0 μm, #3464, CoStar, Cambridge, MA, USA) in serum-free ECM (100 μl). Following H/R and lentiviral treatments, non-migrating cells on the surface of the insert were removed with a cotton swab, while cells that had migrated through the Transwell inserts were washed with PBS and fixed in 4% paraformaldehyde for 20 min. Samples were washed three times with PBS, then stained with crystal violet for 10 min and was washed three times with PBS. Samples were visualized by microscopy. The number of cells was counted in three random fields.

Western Blot Analysis

Lung tissue samples and cell samples were lysed in RIPA buffer (Beyotime Biotechnology Shanghai, China) supplemented with protease inhibitors (Roche Applied Science, Indianapolis, IN, USA) for 30 min on ice. Protein concentrations were measured using a BCA Protein Assay Kit (Beyotime Biotechnology). Protein samples were then separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% skim milk in TBST for 1 h at room temperature, then incubated with primary antibodies against FLRT3 (1:1,000; ab223047, Abcam, Cambridge, MA, USA), RND3 (1:500; sc-53874, Santa Cruz Biotechnology, CA, USA), VE-cadherin (1:1,000; ab205336, Abcam), ZO-1 (1:1,000; ab276131, Abcam), claudin-5 (1:5,000; ab131259, Abcam), occludin (1:1,000; ab216327, Abcam), p-MYPT1 (1:500; ab59203, Abcam), MYPT1 (1:1,000; ab59235, Abcam), RhoA (1:5,000; ab187027, Abcam), p-MCL1 (1:1,000; #4579, Cell Signaling Technology), MCL1 (1:1,000; #94296, Cell Signaling Technology) and LC3I/II (1:1,000; #4108, Cell Signaling Technology) overnight at 4 °C. Membranes were washed three times with TBST, and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies. Protein bands were visualized using an Enhanced Chemiluminescence Kit.

RhoA-GTP (Rhotekin-RBD Pull-Down) Assay

GTP-bound RhoA was measured using a RhoA Activation Assay Kit (Merck Millipore, Billerica, USA). Briefly, equal amounts (1,000 µg) of each cell lysate were incubated with 30 µg GST-Rhotekin Rho-binding domain coupled to glutathione-agarose beads for 45 min. The beads were washed three times with washing buffer. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. RhoA was detected by western blot using an anti-RhoA antibody (1:1,000; ab187027, Abcam).

GST Pulldown Assay

His-FLRT3 (ProteinTech, Wuhan, China) and GST-RND3 (ProteinTech) fusion proteins were mixed in GST-binding buffer for 12 h at 4 °C. Then, anti-His or anti-GST beads were added to the fusion proteins and incubated for 4 h. The beads were washed three times and eluted proteins were subjected to western blot analysis.

Co-immunoprecipitation (Co-IP)

Protein was extracted using lysis buffer, and incubated with primary antibodies against FLRT3 (1:100; #3462, Cell Signaling Technology), RND3 (1:100; sc-53874, Santa Cruz Biotechnology) and IgG at 4 °C overnight. The following day, samples were incubated with protein Dynabeads (50 μl; Thermo Fisher Scientific) for 6 h at 4 °C. After washing three times with RIPA buffer, IP lysates (20 μl) were added to 2× loading buffer, boiled, and subjected to western blot analysis.

F-actin Staining

Changes in cytoskeletal structure were monitored by immunofluorescence staining of F-actin. Briefly, HPMECs were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, then stained with fluorescein isothiocyanate (FITC)-conjugated phalloidin (Molecular Probes, Eugene, OR, USA). After washing with PBST four times, cells were visualized using FluorSave reagent (Calbiochem, Billenica, MA, USA) with a fluorescent microscope (Olympus).

Real-Time Quantitative Reverse Transcription PCR (qRT-PCR)

RNA was extracted from lung tissue and HPMECs using the RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Valencia, CA, USA). cDNA was generated and qRT-PCR was performed using the Quantitect SYBR Green RT-PCR kit with the ABI 7300 real time PCR system (Applied Biosystems, Foster City, CA, USA). The following primers were used: human FLRT3: 5′-CGACAGCAGGACAAGCTCA-3′/5′-TCAGAACCCAGGAAGACGAGA-3′; mouse FLRT3: 5′-AGTCAACTGGATGCTCTCGC-3′/5′-GTAAAAACCCTCCCCCGTCT-3′.

Immunofluorescence Microscopy

Lung lobes were removed from mice and placed in 4% paraformaldehyde in PBS at 4 °C overnight. Fixed lungs were paraffin embedded, and 5-µm sections were obtained. Serial lung sections were subjected to dewaxing followed by antigen retrieval (Antigen Unmasking Solution, Vector Laboratories, Burlingame, CA, USA) and blocking for 1 h in 5% normal goat serum, 0.2% Triton X-100, and 0.05% fish skin gelatin. Lung sections were stained with anti-FLRT3 (1:100; ab223047 Abcam) and anti-CD31 (1:100; ab76533, Abcam) antibodies at 4 °C overnight.

HPMECs were washed in PBS, fixed in 10% neutral-buffered formalin for 15 min at 37 °C, then permeabilized with Target Retrieval Solution (Dako, Carpinteria, CA, USA) for 10 min at 95 °C. After cooling, samples were blocked with 1% BSA in PBS containing 0.05% Tween-20 (PBST) at 37 °C for 1 h, then incubated with anti-FLRT3 (1:100; ab223047, Abcam), anti-VE-cadherin (1:1,000; ab205336, Abcam) and anti-claudin-5 (1:500; ab131259, Abcam) antibodies for one hour at 37 °C. Next, samples were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:50, Jackson ImmunoResearch, West Grove, PA, USA) at 4 °C overnight. Finally, samples were mounted using Vectashield Mounting Medium with DAPI (Vector Laboratories) and examined by fluorescence microscopy (Olympus). Images were captured and analyzed using cellSens image analysis software (Olympus).

Statistical Analysis

Data are presented as mean ± SD. An unpaired Student’s t test was used to analyze statistically significant differences among two groups. One‐way ANOVA and Tukey’s multiple comparisons test were used to analyze statistically significant differences among multiple groups. Two‐way ANOVA with Tukey’s post hoc test was used to analyze multiple groups with two categorical variables. Statistical analyses were performed using GraphPad Prism Software version 8.0 (San Diego, CA, USA). P-values < 0.05 were considered to be statistically significant.