Hyperoxia-induced lung microvascular injury is mediated by endothelial cell-derived WNT5A

Abstract

Background

Bronchopulmonary dysplasia (BPD) is a common complication in preterm infants, with dysmorphic microvascular development being a key factor in the pathogenesis of BPD. WNT5A plays a role in angiogenesis and vascular development and is closely associated with lung disease. This study aimed to investigate the role and underlying mechanism of WNT5A in hyperoxia-induced pulmonary microvascular impairment in BPD.

Methods

A hyperoxia exposure mouse model (85% oxygen for 14 days) simulating preterm BPD was used, and was sampled on postnatal day (P) 3, 7, and 14. Western blot (WB) was used to detect the expression of endothelial cell markers von Willebrand factor (vWF) and WNT5A. Mouse pulmonary microvascular endothelial cells (PMVECs) and human umbilical vein endothelial cells (HUVECs) were cultured in 85% oxygen for 48 hours. WNT5A expression and secretion were measured through WB and enzyme-linked immunosorbent assay (ELISA). WNT5A inhibitors or recombinant WNT5A (rWNT5A) were co-cultured with cells, and the cell function was assessed. Total RNA was extracted from PMVECs, followed by RNA sequencing.

Results

WNT5A expression was decreased in the lungs of hyperoxia-exposed mice at P7. Consistently, in vitro hyperoxia exposure reduced WNT5A expression and its downstream effector CaMKIIγ in endothelial cells compared with normoxia-exposed controls. Both WNT5A inhibition and rWNT5A changed the migration and tube formation functions of HUVECs in normoxic and hyperoxic conditions. RNA sequencing revealed differentially expressed genes (DEGs), and suggested enrichment in the ErbB, MAPK, and Hippo pathways in the hyperoxia group.

Conclusions

Hyperoxia decreased the WNT5A expression and impaired endothelial cell function, potentially through WNT5A-CaMKIIγ.

Highlight box.

Key findings

• Hyperoxia decreased the WNT5A expression and impaired endothelial cell function, potentially through WNT5A-CaMKIIγ.

What is known and what is new?

• WNT5A deficiency is associated with abnormal distal airway branching, impaired pulmonary microvascular development, and alveolar simplification. Previous studies have demonstrated that WNT5A regulates endothelial cell proliferation and migration in vivo, as well as pericyte function in vivo and in vitro, through non-canonical signaling pathways.

• WNT5A expression was decreased in the lungs of hyperoxia-exposed mice at postnatal day 7. Consistently, in vitro hyperoxia exposure reduced WNT5A expression and its downstream effector CaMKIIγ in endothelial cells compared with normoxia-exposed controls, leading to impaired endothelial cell function. RNA sequencing further confirmed the downregulation of the WNT5A-CaMKIIγ pathway in hyperoxia-exposed endothelial cells and revealed enrichment of the ErbB, MAPK, and Hippo signaling pathways.

What is the implication, and what should change now?

• Microvascular injury in the lungs of hyperoxia-exposed mice may be attributed to decreased WNT5A signaling via the WNT5A-CaMKIIγ pathway. Investigating the role of the WNT5A-CaMKIIγ axis in hyperoxia-induced endothelial dysfunction, as well as its crosstalk with ErbB, MAPK, and Hippo signaling pathways, may provide new insights into the mechanisms underlying microvascular impairment in bronchopulmonary dysplasia.

Introduction

Background

Bronchopulmonary dysplasia (BPD) is a chronic lung disease commonly observed in preterm infants, characterized by alveolar simplification, impaired pulmonary vascular development, and persistent pulmonary inflammation (1). With advancements in neonatal care, the phenotype of BPD has evolved from old BPD characterized by pulmonary fibrosis and cystic changes, to new BPD marked by simplified alveoli and impaired pulmonary vascular development (2). During normal lung development, pulmonary vascular formation occurs in parallel with alveolarization, and proper vascularization is essential for normal alveolar maturation (3). The vascular hypothesis proposed that impaired microvascular development might contribute to BPD pathogenesis (4). In BPD patients, dysmorphic pulmonary microvasculature was evident, characterized by reduced vessel growth and dilated capillaries embedded within thickened alveolar septa, lacking a well-organized vascular network (4). These structural alterations contributed to pulmonary hemodynamic disturbances, increasing the risk of pulmonary hypertension (PH) and further aggravating disease progression (5,6).

WNT signaling pathway is primarily classified into the canonical (β-catenin-dependent) pathway and the non-canonical (β-catenin-independent) pathway (7). WNT5A serves as a predominant non-canonical WNT ligand, activates downstream signaling through the planar cell polarity (PCP) pathway and the WNT/Ca²⁺ pathway, thereby triggering cytoskeleton rearrangement and regulating cell fate and migration (8,9). Previous studies revealed the crucial role of WNT5A in lung development. Both deletion and overexpression of Wnt5a led to aberrant lung development, including reduced branching, dilation of distal airways, impaired pulmonary microvascular system, and thickened septal walls (10,11). These alternations might be associated with WNT5A regulation of apoptosis and transdifferentiation of alveolar type 2 cells (AT2) (12-14), microvascular-net formation and endothelial cell migration (15-18), as well as smooth muscle cells (SMCs) and myofibroblasts function (19,20). However, the involvement of the WNT5A signaling pathway in BPD remained controversial.

During normal lung development in rats, Wnt5a mRNA expression exhibited dynamic changes from embryonic day 18 (E18) to postnatal day 21 (P21), while hyperoxia exposure reduced Wnt5a mRNA expression in P6 rat lungs (19). Notably, postnatal Wnt5a conditional knockout mice exhibited disrupted alveolarization with a BPD-like phenotype, and decreased WNT5A mRNA expression has also been detected in lung tissues from human BPD patients (21,22). Nevertheless, mesenchymal WNT5A upregulation during hyperoxic injury also contributed to alveolar simplification and interstitial thickening resembling the BPD phenotype (23). Our previous studies demonstrated that mesenchymal stem cell-derived extracellular vesicles ameliorated transdifferentiation of AT2 in hyperoxia-induced BPD rats by downregulating pulmonary WNT5A expression (24).

Therefore, WNT5A is involved in airway and vascular development and is closely associated with BPD pathogenesis. However, the expression changes of Wnt5a in hyperoxia-exposed BPD mouse models remained unclear, and the underlying mechanisms by which WNT5A regulates hyperoxia-induced pulmonary microvascular endothelial injury remain to be elucidated. This study aims to investigate the role of WNT5A in hyperoxia-induced pulmonary vascular injury and its potential molecular mechanisms, providing new theoretical insights for improving pulmonary vascular function in BPD patients. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1-2699/rc).

Methods

Animal and hyperoxia exposure

All animal procedures were approved by the Fudan University Animal Care and Use Committee (project license No. 2025-EKYY-138) and were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. A study protocol was prepared before the study without registration. Healthy C57BL/6J pregnant mice in this study were sourced from GemPharmatech Co. Ltd. (Nanjing, China). Each independent experiment included three pregnant dams with synchronized gestational ages (within a 1-day interval). To minimize oxygen-related maternal toxicity, dams were alternated daily throughout the exposure period. Immediately after term delivery, neonatal mice were allocated to either normoxic or hyperoxic conditions (n=12 per group). Control animals were maintained under room air (RA), whereas the experimental group was housed in a chamber containing 85% oxygen. Carbon dioxide levels were regulated at 0.5% during exposure. The chamber was opened for approximately 15 minutes per day to allow routine replacement of food and water. Neonatal mice of both sexes were used in the experiments. No specific inclusion or exclusion criteria were set for the animals in this study. All animals were included as long as they met the general requirements for the experiment. Animals that exhibited developmental abnormalities, such as body weight or body length significantly deviating from the rest of the group during the experiment, were excluded from the analysis.

Lung sampling and histology

Lung tissues were harvested at P3, P7, and P14. Mice were anesthetized with inhaled isoflurane prior to thoracic cavity exposure. Pulmonary circulation was cleared by right ventricular perfusion with ice-cold phosphate-buffered saline (PBS). For downstream analyses, the right lung lobes were collected and immediately stored at −80 ℃. In contrast, the left lung was inflated in situ with 4% paraformaldehyde (PFA) and subsequently immersed in the same fixative overnight to allow adequate fixation before histological processing. Hematoxylin-eosin (HE) staining was performed by Wuhan Servicebio Technology Laboratory. Radical alveolar counts (RAC) and mean linear intercept (MLI) were calculated with 6 random fields per group to assess the degree of alveolarization (24).

Cell extraction and cell culture

Healthy C57BL/6J pregnant mice were purchased from GemPharmatech Co. Ltd. (Nanjing, China). Pulmonary microvascular endothelial cells (PMVECs) were isolated from the lungs of healthy 7-day-old mice. Briefly, mice were anesthetized with isoflurane (RWD, Shenzhen, China), and the body surface (except the head) was immersed in 75% ethanol (Biosharp, Beijing, China) for 5 min for disinfection, after which the mice were positioned and fixed on a biosafety cabinet. The thoracic cavity was opened to expose the heart, and the ventricles were perfused with Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) containing 90 U/mL heparin (Biosharp) and penicillin-streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin; Gibco).

The lungs were then excised and immersed in DMEM, and dissociated with 0.25% trypsin (Gibco) for 5 minutes. The peripheral lung tissue was cut into approximately 1–2 mm pieces. The tissue pieces were transferred to a 35-mm dish containing fetal bovine serum (FBS) (Gibco) using sterile ophthalmic forceps and incubated for 45 min. Subsequently, the tissue pieces were evenly placed onto 1% gelatin-coated (Solarbio, Beijing, China) 6-well plates or glass coverslips (for further immunofluorescence), which were positioned at an angle in the incubator for 1 h to facilitate firmer attachment. Thereafter, 500 µL of endothelial cell medium (ECM; ScienCell, Carlsbad, CA, USA) was added to each well, and the cultures were maintained at 37 ℃ in a 2% O₂ incubator. An additional 500 µL of ECM was added after 24 h. After 60 h, the tissue explants were removed, and the medium was replaced. Subsequently, half to two-thirds of the medium was refreshed every 2–3 days. We also scraped independent fibroblast colonies and sparsely distributed fibroblasts under the microscope. After approximately 7 days, the cells reached about 90% confluence. For passaging, 0.25% trypsin was added for digestion for 2–3 min, and DMEM containing 10% FBS was added to terminate digestion. The cells were gently pipetted repeatedly to obtain a single-cell suspension, which was then collected into a centrifuge tube and centrifuged at 1,000 r/min for 5 min. The supernatant was discarded, and the cell pellet was resuspended in ECM medium before being returned to the incubator for further culture. Subsequent passages were performed at a ratio of 1:2.

Human umbilical vein endothelial cells (HUVECs) were purchased from FuHeng Cell Center (FH1122, Shanghai, China). The PMVECs and HUVECs were cultured with an oxygen concentration of 21% or 85% O₂ for 48 h, 100 ng/mL recombinant WNT5A (rWNT5A; R&D Systems, Minneapolis, MN, USA) or 60 mmol/L Box5 (MCE, Monmouth Junction, NJ, USA) was added to the cells according to the experiment group.

Immunofluorescence assay

Immunofluorescence staining was performed on PMVECs grown on glass coverslips. Cells were fixed in 4% PFA and washed gently with PBS before membrane permeabilization using 0.1% Triton X-100. Blocking was carried out with 10% bovine serum albumin (BSA) to reduce nonspecific background signals. Cells were then incubated with mouse anti-CD31 antibody (1:20, sc-20071, Santa Cruz, Dallas, TX, USA) overnight at 4 ℃, washed, and incubated with Cy3-conjugated secondary antibodies (AB_2890957, Proteintech, Wuhan, China). Finally, nuclei were counterstained with 4',6‑diamidino‑2‑phenylindole (DAPI).

Wound-healing test

We used a sterile pipette tip to make uniform vertical scratches in the HUVEC cell monolayer and immediately washed the floating cells in the culture medium with PBS (Biosharp) to prevent them from affecting the experimental results. We supplemented the diluted culture medium to ensure that the cells can continue to grow and migrate. At 0 and 24 h after the scratch, we used a microscope to observe and photograph the cell migration. We ensured that the position of each shot is consistent for later analysis. Finally, we used ImageJ to adjust the contrast to better distinguish the cell-free area from the cell-covered region. We measured the area of the wound at 0 h and at the 24 h time points. The migrated area was calculated as the difference between these two values to quantify the degree of cell migration.

Transwell experiment

We digested and resuspended the HUVEC cells and adjusted the cell density to [1–10]×10⁵/mL. We added 600 µL ECM (ScienCell) culture medium to the bottom of the 24-well plate, placed the chamber (Corning, Corning, NY, USA) in the 24-well plate, and added 200 µL cell suspension to the upper chamber. We rinsed the cells with PBS (Biosharp) and fixed them with methanol (Biosharp). After staining the cells with 0.1% crystal violet stain (Solarbio), 5 fields of view were randomly selected under a 20× microscope to take pictures, and the number of migrated cells was counted using ImageJ.

Tube formation experiment

We adjusted the digested and resuspended the HUVEC cells to a density of 1.5×10⁵ cells/500 µL ECM (ScienCell) per well and seeded them in a 24-well plate pre-embedded with Matrigel (Corning). Then, we took pictures at 2, 4, and 6 h, and used ImageJ to count the number of junctions segments, total segments length, and meshes.

Western blot (WB)

Protein lysis from mouse lung, HUVEC, and PMVEC were separated by 10–12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were subsequently transferred onto 0.45 µm NC membranes (Millipore, USA). The membranes were blocked with 5% BSA (Beyotime, Shanghai, China) in TBST for 2 h at room temperature. After blocking, membranes were incubated overnight at 4 ℃ with primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AB_3102089; MCE), WNT5A (ab227229; Abcam, UK), and CaMKIIγ (AB_2068213; Proteintech). Following primary antibody incubation, membranes were treated with horseradish peroxidase-conjugated secondary antibodies (HY-P83652; MCE). Protein bands were detected using enhanced chemiluminescence reagents (Beyotime).

Enzyme-linked immunosorbent assay (ELISA)

ELISA was performed using a commercial kit (mlbio, Shanghai, China) according to the manufacturer’s instructions. Supernatants from both PMVECs and HUVECs were clarified by centrifugation at 3,000 rpm for 20 min prior to analysis. Samples were added to the wells, and absorbance at 450 nm was measured using a microplate reader.

RNA sequencing

PMVECs for RNA sequencing were purchased from IMMOCELL (IMP-M027, Xiamen, China). We set three experimental groups for RNA sequencing, involving PMVECs in RA, hyperoxic PMVECs (HO), and hyperoxic PMVECs with rWNT5A (HO-rWNT5A). Three replicates were included in each group. RNA from each sample was purified using the RNAiso Plus kit (TAKARA, Kusatsu, Shiga, Japan), and its quality and quantity were assessed via gel electrophoresis and Qubit (Thermo, Waltham, MA, USA). For RNA sequencing, libraries were prepared from 1 µg of total RNA using the VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina^®. Sequencing was performed as 150-bp paired-end reads on the Illumina NovaSeq X Plus platform by Genergy Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions. The raw data of all samples had been submitted to the Sequence Read Archive at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/sra) under accession number GSE320519.

Data analysis

Sequencing data were first trimmed with Skewer, followed by quality assessment using FastQC v0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The read length was 2×150 bp. Clean reads were aligned to the Human genome hg38 using STAR. StringTie. The expression of the transcript was calculated by FPKM (fragments per kilobase of exon model per million mapped reads) using Perl.

To identify genes differentially expressed between case and control samples, DESeq2 in R was applied using the likelihood ratio test. Genes with adjusted P values ≤0.05 and at least a two-fold change were retained for downstream analyses. When normalized read counts were near zero, log₂ transformation with a pseudocount of 1 was performed. Heatmaps illustrating gene expression were generated with the pheatmap package. Functional and pathway enrichment analyses of differentially expressed genes (DEGs) were conducted using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and pathways including at least two associated genes with P<0.05 were considered significant. Gene set enrichment analysis (GSEA) was performed to evaluate whether predefined gene sets exhibited coordinated expression differences between two biological conditions. Genes were ordered according to their differential expression levels, and enrichment of specific gene sets at the extremes of the ranked list was assessed. This approach allows detection of biologically meaningful yet modest expression alterations. The local GSEA software was applied, and Hallmark as well as KEGG gene sets were analyzed separately.

Statistical analysis

Statistical analysis was performed in the GraphPad Prism software. Data were presented as the mean ± standard deviation (SD). For comparisons between two independent groups with normally distributed data (tested by Shapiro-Wilk test), an unpaired t-test was used. For non-normally distributed independent samples, the Mann-Whitney U test was applied. ELISA data were analyzed using four-parameter logistic curve fitting. Statistical significance was defined as P<0.05.

Results

WNT5A expression was decreased in hyperoxia-exposed mouse lung and endothelial cells

Hyperoxia led to simplified alveolar development and impaired vascular formation (Figure 1A). MLI was increased at P7 and P14 BPD mouse model, while RAC remarkablely decreased at P14 (Figure 1B,1C). The vascular endothelial markers, including von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF), were downregulated (Figure 1D,1E). Compared with the normal condition, the expression of WNT5A decreased in hyperoxia-exposed mouse lungs at P7 (Figure 1F,1G). The same results were shown in vitro PMVECs model (Figure 2). We isolated PMVECs from lung tissues of healthy P7 mice via tissue block and endothelial cell culture medium. PMVECs started migrating from tissue explants after 24 hours of culture (Figure 2B). The expression of the PMVECs marker CD31 was confirmed by immunofluorescence (Figure 2A). It is also worth noting that cilia could be seen using a scanning electron microscope (SEM), while plasma membrane vesicles could be identified with a transmission electron microscope (TEM) (Figure 2C,2D). These ultrastructural characteristics were in line with the expected features of PMVECs, supporting the validity of the isolation procedure. Primary PMVECs were exposed to hyperoxia for 48 h, then harvested for WB and ELISA. Exposure to hyperoxia significantly reduced the expression of WNT5A in PMVECs, while the same results were shown in HUVECs exposed to 48 h hyperoxia (Figure 2E-2J). CaMKIIγ was also decreased in PMVECs and HUVECs under hyperoxia exposure (Figure 2E-2H).

Figure 1.

Hyperoxia exposure resulted in abnormal distal airway branching and impaired pulmonary microvascular development. (A) Representative images of HE-stained mouse lungs under hyperoxia and normoxia exposure (n=6). (B) MLI and (C) RAC measurements. (D,E) Western blot analysis and quantification showing significantly reduced vWF and VEGF in the BPD mouse model (n=3). (F,G) Western blot analysis and quantification showing significantly reduced WNT5A in the BPD mouse model (n=3). Data are shown as mean ± SD. n=6; *, P<0.05; **, P<0.01; ****, P<0.0001. BPD, bronchopulmonary dysplasia; HE, hematoxylin-eosin; HO, hyperoxic PMVECs; MLI, mean linear intercept; PMVEC, pulmonary microvascular endothelial cell; RA, room air; RAC, radical alveolar counts; SD, standard deviation; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.

Figure 2.

Hyperoxia decreased the expression of WNT5A and CaMKIIγ in endothelial cells. (A) Immunofluorescence staining (×20) of PMVECs for CD31 (red) and DAPI nuclear staining (blue). (B) Migration of PMVECs from tissue blocks. (C) SEM images showing cell microvilli (*). (D) TEM image showing plasma membrane vesicles (red arrow). (E,F) Western blot analysis demonstrated significantly reduced WNT5A and CaMKIIγ in (G) PMVECs and (H) HUVECs in hyperoxia exposure compared with normoxia. (I,J) ELISA showing decreased WNT5A levels in cell supernatant in hyperoxia exposure under hyperoxia. Data are shown as mean ± SD. n=3; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ELISA, enzyme-linked immunosorbent assay; HO, hyperoxic PMVECs; HUVEC, human umbilical vein endothelial cell; PMVEC, pulmonary microvascular endothelial cell; RA, room air; SD, standard deviation; SEM, scanning electron microscope; TEM, transmission electron microscope.

Hyperoxia exposure disrupted the migration and angiogenesis of endothelial cells partly through WNT5A-CaMKIIγ signaling pathway

Cell migration and tube formation are two major abilities associated with angiogenesis of endothelial cells. We conducted wound-healing and transwell assays to test the migration of HUVECs. The results showed that hyperoxia caused a decrease in the migration area of HUVECs, and decreased the cells’ ability to cross the transwell chamber membrane (Figure 3A-3D). However, co-culture with 100 ng/mL rWNT5A could partially restore the migration dysfunction caused by hyperoxia, although it could not recover to the level under normoxia (Figure 3A-3D). We cultured HUVECs with Matrigel to investigate their tube formation ability under different conditions. Compared with normoxia, exposure to hyperoxia significantly impaired the tube networks, as evidenced by a decrease in the total number of junctions and segments, the total length of segments, and the number of meshes in each timing (Figure 3E-3I, Figure S1A,S1B). Co-cultured HUVECs with 100 ng/mL rWnt5A in hyperoxia could partly rescued the tube formation ability, showing increased total number and length of junction and segments at the 4-hour time point (Figure 3E-3I).

Figure 3.

rWNT5A partially rescues hyperoxia-induced migration and tube formation dysfunction in HUVECs. (A) Representative images of wound-healing co-culture assays; scale bar: 250 µm. (B) Quantification of cell migration area after 24 h. (C) Representative graphs of transwell assays at 24 h; scale bar: 50 µm. (D) Quantification of the number of cells crossing at 24 h in transwell assays. (E) Representative images of tube formation experiment at 2, 4, and 6 h; scale bar: 100 µm. (F-I) Quantification of junction, segments, total length of segment, and meshes in tube formation experiment at 4 h. Data from 2 and 6 h that did not show statistically significant differences are presented in Figure S1. Data are shown as mean ± SD. n=3; *, P<0.05; **, P<0.01; ****, P<0.0001. HO, hyperoxic PMVECs; HO-rWNT5A, hyperoxic PMVECs with rWNT5A; HUVEC, human umbilical vein endothelial cell; PMVEC, pulmonary microvascular endothelial cell; RA, room air; SD, standard deviation.

Box5 is a WNT5A antagonist that inhibits WNT5A signaling and WNT5A-mediated Ca²⁺ release. In our study, we found that Box5 impaired HUVEC migration and tube formation under both normoxic and hyperoxic conditions (Figure 4). The results showed that 60 µmol/L Box5 could effectively inhibit the release of cellular WNT5A after co-culture for 48 h (Figure 4B). Next, we repeated the wound-healing and transwell assays to explore the effect of Box5 on the migration function of HUVECs under hyperoxia and normoxia. The results of these assays suggested that Box5 inhibited cell migration and decreased the transmembrane ability in normoxic conditions (Figure 4A,4C-4E). When it came to the hyperoxia condition, Box5 would further exacerbate the cell migration and transmembrane ability (Figure 4A,4C-4E). The damage of Box5 to the tube-forming function of HUVECs was more significant than the improvement of rWNT5A. After 2 h of the tube formation assays, it was observed that Box5 significantly reduced the formation of junctions, segments, total length of segments and number of meshes of normoxic HUVECs, and its damaging effect lasted to the end of the observation at 6 h, while significant decrease of the number of junctions, segments and meshes in HUVEC co-cultured with Box5 under hyperoxia exposure was observed at 4 and 6 h (Figure 4F-4J, Figure S1C,S1D).

Figure 4.

Box5 inhibited HUVECs migration and tube formation in hyperoxia and normoxia conditions. (A) Representative images of wound-healing co-culture assays, scale bar: 250 µm. (B) 60 mmol/L Box5 reduced WNT5A expression in HUVECs. (C) Quantification of cell migration area after 24 hours. (D) Representative graphs of transwell assays at 24 h, scale bar: 50 µm. (E) Quantification of the number of cells crossing at 24 h. (F) Representative images of tube formation experiment at 2, 4, and 6 h, scale bar: 100 µm. (G-J) Quantification of junction, segments, total length of segments, and meshes in tube formation experiment at 4 h. Data from 2 and 6 h that did not show statistically significant differences are presented in Figure S1. Data are shown as mean ± SD. n=3; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO, hyperoxic PMVECs; HUVEC, human umbilical vein endothelial cell; PMVEC, pulmonary microvascular endothelial cell; RA, room air; SD, standard deviation.

Therefore, we primarily suggested that hyperoxia inhibited the migration and tube formation of vascular endothelial cells through reduced expression of WNT5A-CaMKIIγ pathway.

Hyperoxia exposure altered the gene expression of PMVECs

We set three experimental groups for RNA sequencing, involving PMVECs in RA, HO, and HO-rWNT5A (Figure 5). Three replicates were included in each group. Through differential expression analysis, 1,459 mRNAs were found to be downregulated and 1,422 upregulated in the HO vs. RA group, while 1,119 mRNAs were downregulated and 1,232 upregulated in the HO vs. HO-rWNT5A group. Nedd9 was the most significantly reduced gene in the HO vs. RA group, and Sptan1 expression was significantly upregulated in the HO vs. HO-rWNT5A group (Figure 5A,5C).

Figure 5.

Volcano plot and heatmap of DEGs. (A) Volcano plot of all DEGs in HO vs. RA. (B) Heatmap of DEGs related to the WNT signaling pathway in the HO vs. RA group. (C) Volcano plot of all DEGs in the HO-rWNT5A vs. HO group. (D) Heatmap of DEGs related to the WNT signaling pathway in the HO-rWNT5A vs. HO group. (E) The Venn diagram showed the overlap genes between two comparison groups, including (F) Camk2g, (G) Camk2a, (H) Nfatc1, (I) Tbl1xr1, (J) Mapk9, (K) Myc, (L) Csnk2a2. (n=3). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. DEG, differentially expressed gene; HO, hyperoxic PMVECs; HO-rWNT5A, hyperoxic PMVECs with rWNT5A; PMVEC, pulmonary microvascular endothelial cell; RA, room air.

According to the KEGG database, DEGs involved in WNT pathway were selected. The results showed that, compared with the RA group, the expression levels of non-canonical Wnt5a and its downstream molecules Camk2g, Camk2a, Mapk9, and Mapk8 in the HO group were significantly reduced in HO group, consistent with the previously reported results. In contrast, the expression of Fzd10 and Myc in the canonical pathway was significantly upregulated (Figure 5B). After co-culture with 100 ng/mL rWNT5A, a significant upregulation of Camk2g, Camk2a, Mapk9, and Rac1 was observed, while a downregulation of Myc was confirmed when compared with HO group (Figure 5D). Seven genes served as DEGs in both groups, including Camk2g, Camk2a, Nfatc1, Tbl1xr1, Mapk9, Myc, and Csnk2a2 (Figure 5E-5L).

Hyperoxia-associated signaling network remodeling

To gain a deeper understanding of biological functions, we performed GO, KEGG and Reactome pathway enrichment analysis of these DEGs. In the GO enrichment analysis, the results of the two comparison groups showed enrichment in cell metabolic process, intracellular organelles, and protein binding (Figure 6A,6B). Compared with the RA group, WNT associated DEGs in HO group were mainly enriched in the formation and closure of the tube, including the neural tubes, epithelial tubes, and blood vessels (Figure S2A). Furthermore, WNT-associated DEGs in HO-rWNT5A group were enriched in mesenchymal development and morphogenesis, nervous system development, embryonic organ development, epithelial tubes and trachea, kidney development when compared with HO group (Figure S2B).

Figure 6.

Enrichment analysis of DEGs. (A,C,E,G) GO, KEGG, Reactome, and GSEA enrichment analyses of all DEGs in the HO vs. RA group. (B,D,F,H) GO, KEGG, Reactome, and GSEA enrichment analyses of all DEGs in the HO-rWNT5A vs. HO group (n=3). BP, biological process; CC, cellular component; DAG, diacylglycerol; DEG, differentially expressed gene; FDR, false discovery rate; GO, Gene Ontology; GSEA, gene set enrichment analysis; HO, hyperoxic PMVECs; HO-rWNT5A, hyperoxic PMVECs with rWNT5A; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; NES, normalized enrichment score; PC, phosphatidylcholine; PMVEC, pulmonary microvascular endothelial cell; RA, room air; rWNT5A, recombinant WNT5A.

The KEGG pathway analysis revealed an increase in the ErbB signaling pathway and MAPK signaling pathway in the two comparison groups (Figure 6C,6D). In addition, the Reactome enrichment analysis indicated the DEGs in HO group were concentrated in processes such as basal body anchoring to the plasma membrane when compared with the RA group, while the HO-rWNT5A group was enriched in integrin signaling pathway when compared with HO group (Figure 6E,6F).

We used the gene set of the canonical WNT pathway in Hallmark for GSEA analysis. The normalized enrichment score (NES) obtained in the HO vs. RA group was 1.35, indicating a higher functional activity and expression levels of canonical WNT pathway in the HO group. However, this conclusion was not significant (P=0.04 and P_adj=0.79, did not meet the requirements of P_adj<0.05 and q value <0.25). This trend might be related to the inhibitory effect on the canonical pathway caused by low expression of WNT5A under hyperoxia. In the HO vs. HO-rWNT5A group, NES was −1.23, indicating a decreased enrichment in the HO-rWNT5A group without significance (P=0.11 and P_adj=0.97) (Figure 6G,6H). Only the Hippo signaling pathway (mmu04390, NES =1.99, P_adj=0.006, q value =0.007) and the signaling pathway regulating pluripotency of stem cell pluripotency (mmu04550, NES =1.90, P_adj=0.02, q value =0.02) in the HO vs. RA group simultaneously met the requirements of P_adj<0.05 and q value <0.25 (Figure S2C,S2D).

Discussion

The present study demonstrated that hyperoxia downregulated WNT5A expression in vivo and in vitro, and that this reduction was associated with impaired migration and tube formation functions. Importantly, supplementation with recombinant WNT5A partially restored endothelial function, while Box5 would further exacerbate the cell migration and transmembrane ability under hyperoxic conditions. These findings suggest that WNT5A may serve as a critical regulatory factor in maintaining pulmonary endothelial homeostasis during hyperoxic stress. Given that impaired pulmonary angiogenesis and vascular dysfunction are central pathological features of BPD, our findings suggest that hyperoxia-induced downregulation of WNT5A may contribute to endothelial dysfunction and disrupted vascular development.

BPD is a serious chronic lung disease in premature infants, which is more likely to develop in premature infants with a gestational age <29 weeks (6). The new BPD is mainly characterized by lung development stagnation with alveolar simplification and pulmonary vascular development disorders, while the lung injury and repair characteristics of pulmonary fibrosis and pulmonary cystic lesions are gradually reduced (25).

The WNT signaling pathway is complex and highly interconnected, functioning through both canonical (β-catenin-dependent) and non-canonical pathways (26,27). Previous studies have shown that canonical WNT signaling is involved in lung development and BPD pathogenesis. Abnormal activation of β-catenin signaling has been reported in children with BPD, and modulation of this pathway can influence disease severity in experimental models (28-31). However, findings regarding β-catenin expression and its downstream targets remain inconsistent across different studies and models (32-34). Long et al. (35) showed that miR-146b-5p activated the WNT canonical signaling pathway by regulating KDM6B, exacerbating hyperoxia-induced inflammation and autophagy of alveolar epithelial cells. Jia (36) found that YAP and WNT3A can increase nuclear β-catenin levels, promote AT2 proliferation and differentiation, and improve the BPD model. In the BPD organoid model, fibroblast β-catenin Y489 has abnormal and persistent phosphorylation, which can be used as one of the indicators for detecting BPD (37). The above studies all show that the canonical WNT signaling pathway is involved in the occurrence and development of BPD but is subject to complex and context-dependent regulation.

WNT5A is a key signaling molecule in the WNT non-canonical pathway and is involved in the occurrence and development of BPD. Studies have shown that WNT5A and ROR mRNA expression is reduced in the lung group of BPD children, and the postnatal Wnt5a conditional knockout mouse model shows interrupted alveoli, showing a phenotype similar to BPD (21,22). In our study, WNT5A expression was reduced in hyperoxia exposure at P7 BPD mouse models. Some studies have also shown that hyperoxic injury promotes mesenchymal Wnt5a expression, leading to alveolar development disorders and interstitial thickening similar to the BPD phenotype (23). Previous studies have shown that umbilical cord mesenchymal exosomes can improve the lung pathological manifestations of hyperoxic injury BPD mouse models by reducing WNT5A expression in the lungs (24). Therefore, the regulation of WNT5A in the hyperoxic BPD mouse model remains controversial, and its role in hyperoxia-induced pulmonary vascular development disorders and downstream mechanisms has not been studied in depth. Future studies could assess WNT5A expression across multiple postnatal stages, both during and after hyperoxic exposure, to better understand its temporal dynamics and functional impact.

In our study, we performed in vitro experiments and RNA-seq analysis of PMVECs to illustrate WNT5A expression and its downstream changes under hyperoxic conditions, aiming to explore the potential relationship between hyperoxia-induced impairment and WNT5A signaling pathway. The results showed that, compared with the normoxic group, the intracellular synthesis and secretion of WNT5A in PMVECs and HUVECs were decreased under hyperoxia, along with its downstream effector CaMKIIγ. Since endothelial cell migration and tube formation are critical for pulmonary microvascular formation, we examined these processes in HUVECs. Hyperoxia inhibited both migration and tube formation, whereas co-treatment with recombinant WNT5A partially rescued these defects. Box5, a WNT5A-Ca²⁺ antagonist, further impaired endothelial migration and tube formation and exacerbated the effects of hyperoxia. These findings were consistent with previous studies showing that WNT5A antibodies, WNT5A siRNA, and CaMKII inhibitors inhibit HUVEC proliferation and migration (15). Collectively, these results suggest that hyperoxia may reduce endothelial cell migration and tube formation via the WNT5A-CaMKIIγ signaling pathway.

RNA-Seq differential gene analysis of hyperoxic and normoxic PMVECs revealed that nedd9 was significantly reduced under hyperoxia. NEDD9 is a multifunctional adaptor protein. WNT5A can act as an upstream regulator, influencing NEDD9 transport to focal adhesions and thereby modulating cell migration, while NEDD9 itself can regulate CDC42 and contribute to migration function (38,39). Studies have shown that pulmonary vascular development depends on communication between PMVECs and pericytes via the WNT5A-CDC42 signaling pathway (16,40,41). Analysis of WNT-associated-DEGs indicated that, compared with the normoxic group, Wnt5a and its downstream targets Camk2g, Camk2a, Mapk9, and Mapk8 were significantly reduced under hyperoxia. Differential gene analysis of hyperoxia and hyperoxia rWNT5a co-culture groups revealed that Sptan1 expression was significantly upregulated in the co-culture group. SPTAN1 is a filamentous cytoskeleton protein that stabilizes the plasma membrane and organelles, participates in cell adhesion and migration, mediates cell-to-cell contact, and regulates apoptosis (42). Currently, research on the interaction between SPTAN1 and WNT5A is lacking. Moreover, compared with the hyperoxia group, the non-classical pathways Camk2g, Camk2a, Mapk9, and Rac1 were significantly upregulated in the co-culture group. These differential expression results further support the notion that hyperoxia-induced endothelial cell dysfunction may be mediated via the WNT5A-CaMKIIγ signaling pathway.

KEGG enrichment analysis showed that the ErbB signaling pathway and MAPK signaling pathway were enriched in the two comparison groups. ERBB4 gene knockout mice exhibit abnormal lung function and structural alterations, including airway hyperresponsiveness, alveolar simplification, reduced expression of pulmonary surfactant protein D, and chronic inflammation (43). These findings suggest that the ErbB signaling pathway plays an important role in lung development and homeostasis. In addition, the MAPK signaling pathway has been identified as a potential regulatory pathway through which various drugs or small molecules exert therapeutic effects on BPD (44-46), highlighting its relevance in the pathogenesis and treatment of BPD.

Reactome enrichment analysis revealed that the DEGs in the HO vs. RA group were mainly enriched in processes including basal body anchoring to the plasma membrane, AKT-mediated phosphorylation of cytoplasmic target proteins, and APC phosphorylation. Basal body anchoring to the plasma membrane is closely associated with ciliogenesis and cell migration (47). APC, a core component of the β-catenin destruction complex, plays a critical role in the canonical WNT signaling pathway. Previous studies have demonstrated that WNT5A can modulate the PI3K/AKT/PTEN signaling axis, thereby influencing cell proliferation and migration (48). Reactome enrichment analysis showed that the DEGs in the HO-rWnt5a vs. HO group were mainly enriched in processes, such as integrin signaling, cell-extracellular matrix interactions, and cell cycle regulation. Among these, integrin signaling is known to interact with the WNT5A signaling pathway and participate in regulating functions including cell proliferation, migration, invasion and adhesion (49,50).

In the GSEA analysis of the HO vs. RA group, the Hippo signaling pathway was more active in the hyperoxia group. Previous studies have shown that modulation of the Hippo signaling pathway can improve angiogenesis and endothelial cell function (51), suggesting its involvement in vascular development and repair. Moreover, evidence from β-catenin knockout mice indicates that the canonical WNT signaling pathway interacts with the Hippo pathway to exert coordinated biological effects (52). YAP/TAZ are the major transcriptional co-activators of the Hippo pathway. Jia (36) demonstrated that YAP plays a regulatory role in AT2 cell transdifferentiation in a BPD model. These findings collectively suggest potential crosstalk between WNT and Hippo signaling in the pathogenesis of BPD. However, the specific role of Hippo signaling in pulmonary vascular developmental disorders in the BPD model remains unclear and warrants further investigation.

There are several limitations in the current study. The animal models and cell lines used may limit the generalizability of the findings. In addition, WNT5A expression was assessed primarily at a single postnatal time point (PN7), which may not fully capture its dynamic role during lung development under hyperoxic conditions or provide a comprehensive view of the repair phase following injury. Furthermore, RNA-seq was performed only on isolated cells, which limits the assessment of tissue-level interactions. Future studies incorporating whole lung samples could provide a more comprehensive view of transcriptional changes.

Conclusions

WNT5A expression was decreased in the lungs of hyperoxia-exposed mice at P7. Consistently, in vitro hyperoxia exposure reduced WNT5A expression and its downstream effector CaMKIIγ in endothelial cells compared with normoxia-exposed controls, leading to impaired endothelial cell function. RNA sequencing further confirmed the downregulation of the WNT5A-CaMKIIγ pathway in hyperoxia-exposed endothelial cells and revealed enrichment of the ErbB, MAPK, and Hippo signaling pathways.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal procedures were approved by the Fudan University Animal Care and Use Committee (project license No. 2025-EKYY-138) and were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Data Sharing Statement

Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1-2699/dss

jtd-18-04-341-dss.pdf ^{(162.3KB, pdf)}

DOI: 10.21037/jtd-2025-1-2699