TGF-alpha/EGFR signalling mediates retinoic acid-induced lung repair

TGF-alpha/EGFR signalling mediates retinoic acid-induced lung repair | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article TGF-alpha/EGFR signalling mediates retinoic acid-induced lung repair Sek-Shir Cheong, Chunyu Yan, Róisín Mongey, David Chambers, Mark Griffiths, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7704784/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Lung repair involves coordination of multicellular processes, including endothelial angiogenesis and epithelial repopulation. Retinoic acid (RA) signalling is crucial for lung development, homeostasis, and repair, however, the mechanisms through which RA drives repair are still unknown. It has previously been shown that RA has no direct effects on repair of alveolar epithelium, yet in animal studies, RA induces alveolar regeneration. Here we show that RA-induces endothelial angiogenesis which elicits paracrine effects on alveolar epithelial cells to drive repair. Transcriptomic profiling of RA-treated HPMECs undergoing angiogenesis revealed enrichment of wound healing pathways and subsequent in-silico analysis identified several secreted factors as potential mediators of paracrine pro-repair effects on the epithelium. Scratch assays demonstrated that of these secreted factors, only TGFα promoted wound healing in alveolar epithelial A549 and primary human alveolar type 2 (hAT2) cells. Further investigation determined that TGFα promoted epithelial repair by enhancing cell migration through EGFR activation, without affecting proliferation or apoptosis. Our findings identify the TGFα/EGFR axis as a key mediator of RA-induced alveolar repair and provide a potential novel therapeutic avenue to enhance alveolar regeneration. Biological sciences/Cancer Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Molecular biology TGF-alpha retinoic acid EGFR signalling angiogenesis alveolar repair pulmonary microvascular endothelial cells lung repair cell migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction A common denominator in many lung diseases is insufficient functional alveolar surface area for gas-exchange. The root cause of this may differ, for example in emphysema, a pathology associated with chronic obstructive pulmonary disease (COPD), the alveoli are destroyed due to repeated insults to the lungs causing tissue destruction. In the case of bronchopulmonary dysplasia, a disease of prematurity, infants fail to form an appropriate number of alveoli, leading to compromised gas-exchange requiring oxygen support and life-long increased susceptibility to other lung diseases. Currently there are no treatments capable of restoring damaged lung parenchyma but one promising avenue through which this might be achieved, is to develop a regenerative medicine treatment to repair or (re)grow alveoli ( 1 , 2 ). The lungs possess significant ability to regenerate and repair following injury, and we are beginning to understand the underlying cellular mechanisms governing these processes ( 3 ). For example, there are populations of tissue stem or progenitor cells that reside at different anatomical locations in the lungs which can become activated upon lung injury and contribute to regenerating the tissue ( 4 , 5 ). In addition, other cell behaviours such as cell migration, proliferation, and cell plasticity contribute to repair of lung tissue ( 6 ). Pharmacological-based modification of alveolar repair has begun to show real therapeutic potential for treating parenchymal lung diseases ( 7 ). One possible approach to develop a regenerative medicine treatment is to identify the molecular signals that drive endogenous lung repair/regeneration and harness these to repair damaged lung tissue. However, this requires detailed knowledge about the endogenous pro-repair pathways. Emerging lung pro-repair factors include Wingless-type MMTV integration site family (Wnts), fibroblast growth factors (FGFs), and sonic hedgehog growth factors ( 8 – 10 ). In addition, the Vitamin A metabolite, retinoic acid (RA) plays a critical role in lung development and alveolar regeneration ( 11 , 12 ). To date, research on alveolar repair and regeneration has largely focused on the epithelial population, with comparatively less attention given to the capillary endothelium, despite its equally critical role in gas exchange ( 13 ). RA is a well-known pro-regenerative factor across species, and studies in animal models demonstrated its ability to drive alveolar regeneration ( 14 ). Human studies identified an imbalance between synthesis and breakdown of RA metabolism in emphysematous lungs, and RA signalling was activated in the microvascular endothelium, which stimulates angiogenesis but does not directly induce epithelial repair ( 15 ). In addition, Ding and colleagues showed that endothelial-derived angiocrine signals induce and sustain alveolar regeneration ( 16 ). Capitalising on these research findings, we hypothesised that factors secreted from the microvascular endothelium during RA-induced angiogenesis act as endogenous pro-repair mediators that drive alveolar epithelial repair in a paracrine manner. To test this hypothesis, we conducted microarray analysis of RA-treated lung microvascular endothelial cells undergoing angiogenesis and identified several candidate secreted proteins. We subsequently tested the pro-repair capabilities of these secreted proteins in vitro and found that transforming growth factor-α (TGFα), secreted from the RA-activated microvascular endothelium, promotes repair of the human alveolar epithelium via epidermal growth factor receptor (EGFR). TGFα, one of seven known EGFR ligands, has previously been shown to be important for alveologenesis ( 17 ). These key mechanistic findings advance our understanding of how RA mediates lung repair and provide a foundation for developing evidence-based regenerative strategies to treat chronic lung diseases. Results Increased RA signalling activity following acid injury in mouse Precision-Cut Lung Slices (PCLS) To explore the role of RA signalling pathway in lung repair, we generated mouse PCLS and conducted quantitative real-time PCR (qRT-PCR) to assess the expression of RA pathway components in uninjured controls versus acid-injured PCLS. Acid injury was induced by exposure to 0.1 M HCl for 1 min, and mRNA levels of RA receptors ( Rara, Rarb , and Rarg ), RA synthesis genes ( Aldh1a1, Aldh1a2 , and Aldh1a3 ), and RA degradation genes ( Cyp26a1 , Cyp26b1 , and Cyp26c1 ) were quantified 48 hr post-injury. As shown in Fig. 1 , Rarb and Rarg transcript levels were significantly increased by 4.9-fold ( p = 0.029) and 2.3-fold ( p = 0.029), respectively, following acid injury of mouse PCLS. The RA synthesis gene Aldh1a3 was also significantly upregulated, showing a 2.2-fold increase ( p = 0.029), whereas Aldh1a2 expression decreased to 0.6-fold ( p = 0.029). Additionally, the RA degradation genes Cyp26a1 and Cyp26c1 showed significant upregulation with increases of 2.5-fold ( p = 0.029) and 1.6-fold ( p = 0.029), respectively, compared to uninjured control PCLS. No significant changes were observed for Rara , Aldh1a1 , or Cyp26b1 . Collectively, these data suggest an increased RA signalling activity following acid injury, highlighting a role for RA signalling in the early stages of lung repair. RA stimulates angiogenesis and wound healing in endothelial cells, but not in alveolar epithelial cells Our previous work demonstrated that RA induced angiogenesis in human pulmonary microvascular endothelial cells (HPMECs) ( 15 ). As a prelude to microarray experiments, we replicated the experiment. Consistent with previous findings, we found that tube length, averaging 40,546 µm per field of view with RA treatment, compared to 32,225 µm in the 0.1% vehicle ethanol (EtOH) control ( p = 0.0317) (Figs. 2 A-C). Furthermore, the total number of nodes per field of view increased from 141 in the control to 217 in RA-treated HPMECs ( p = 0.0317) (Fig. 2 D). To assess whether RA also modulates wound healing capacity in other endothelial cell types, scratch wound assays were performed on human umbilical vein endothelial cells (HUVECs) treated with varying concentrations of RA. Compared to vehicle EtOH-treated controls, RA significantly enhanced wound healing in a dose-dependent manner. The most pronounced effect was observed at 10 µM RA, resulting in an 80.1% wound healing compared to 38.8% in the control ( p = 0.0002). Treatment with 5 µM RA led to 67.2% wound closure ( p = 0.0341), while 3 µM RA increased closure to 61.0%, though this was not statistically significant. HUVECs cultured in complete endothelial growth media (EGM) with 3% foetal calf serum (FCS), serving as a positive control, exhibited high wound healing capacity (86.1%) (Figs. 2 E and F). However, higher RA concentrations (20 µM and 50 µM) were toxic, leading to cell death after 19 hours of incubation (Fig. 2 G). We also confirmed that consistent with previous findings ( 15 ), direct treatment of the alveolar epithelial cell line A549 with RA did not induce wound healing (Fig. 2 H). A549 cells cultured in DMEM supplemented with 10% foetal bovine serum (FBS) served as a positive control, demonstrating nearly complete wound closure after 19 hours (Fig. 2 H). Transcriptomic profiling of RA-induced responses in HPMECs To investigate the mechanisms underlying RA-induced angiogenesis in HPMECs, transcriptomic profiling was performed to examine gene expression changes during this process. Time-lapse imaging of angiogenesis was first conducted to determine critical time points for further analysis. As shown in Supplementary Video S1, RA-treated HPMECs rapidly migrated and reorganised into tube-like structures within 40 minutes of seeding on Matrigel, whereas vehicle-treated (EtOH) controls remained randomly distributed at the same time point (Video S2). By 60 minutes, clear differences in tube formation were evident between control and RA-treated cells (Videos S1 and S2). By 200 minutes, extensive tube networks had formed in RA-treated HPMECs. Based on these observations, RNA was extracted at 40 min, when tube initiation was first apparent, and at 4 hr, when significant networks were evident, for whole-genome expression analysis comparing EtOH control versus RA-treated HPMECs (Fig. 3 A). Microarray analysis identified 962 differentially expressed genes (DEGs) at 40 minutes and 545 DEGs at 4 hours post-treatment between control and RA-treated HPMECs [fold change > 1.5 or < -1.5, false detection rate (FDR)-adjusted p-value < 0.05] (Supplementary Tables S1 and S2). Notably, volcano plot analysis highlighted key components of the RA signalling pathway, including RARB (retinoic acid receptor beta), DHRS3 (dehydrogenase/reductase 3), NRIP1 (nuclear receptor interacting protein 1), and RDH10 (retinol dehydrogenase 10), among the most upregulated genes at 4 hours post-RA treatment (Figs. 3 B and C). In contrast, none of the top genes upregulated at 40 minutes were related to RA signalling (Figure S1 ). RA is the primary ligand for RAR-RXR heterodimers, which bind to retinoic acid response elements (RAREs) to initiate transcription of RA target genes ( 18 ). To further investigate the response of HPMECs to RA, the top 15 DEGs at both the 40-minute and 4-hour time points were analysed to determine whether these genes contained a RARE motif (Supplementary Tables S3 and S4). In silico prediction of RARE DR5 sequences was performed using oPOSSUM3, and results were cross-referenced with previously published RARE-containing genes ( 19 , 20 ). Interestingly, the top 15 upregulated genes at 4 hours showed significant enrichment for RARE-containing genes (47%; 7 out of 15 genes), including DHRS3, RARB, NRIP1, HIC1, HOXA3, HOXA5, and ARHGAP18 (Table S3 ). In contrast, none of the top 15 upregulated genes at 40 minutes contained RARE sequences (Table S4 ). These findings strongly suggest that a cellular response to exogenous RA has occurred by 4 hours post-treatment, leading us to focus our subsequent gene enrichment and functional analyses on the 4-hour dataset. Previous studies have shown that RA induces the upregulation of vascular endothelial growth factor A ( VEGFA ) ( 15 ). Consistent with this, our findings showed increased VEGFA expression at 40 minutes and upregulation of vascular endothelial growth factor receptor 1 ( VEGFR1 or FLT1 ) at 4 hours post-RA treatment. No changes were found in other genes involved in VEGF signalling, including VEGFB , VEGFC, FLT4 (VEGFR3), KDR (VEGFR2) , neuropilin-1 ( NRP1 ), and neuropilin-2 ( NRP2 ) (Figure S2 ). Enriched GO terms and pathways in RA-treated HPMECs Since cellular responses to RA have occurred by 4 hours post-treatment (Figs. 3 B and C, and Table S3 ), we performed gene set enrichment analysis (GSEA) on DEGs from this time point using Panther and Cytoscape. Several key biological processes were significantly enriched in RA-treated HPMECs. The most prominent processes included regulation of cell adhesion, developmental processes, and actin filament organisation, all highly significant after FDR adjustment. Additional enriched processes, including epithelial cell proliferation, differentiation, cell motility, response to wounding, and tube morphogenesis, pointed to a robust cellular response to RA (Fig. 4 A). Pathway enrichment analysis further confirmed activation of RA signalling in RA-treated HPMECs. Pathways directly related to RA, including retinoic acid signalling and RA biosynthesis, were significantly enriched at 4 hours post-treatment (Fig. 4 B). Additionally, pathways such as interferon signalling and CCKR signalling suggest that RA also modulates immune responses. Notably, the EGF receptor (EGFR) signalling pathway, which has been implicated in lung development( 21 ) and various chronic pulmonary diseases, including lung fibrosis, COPD, asthma, and lung cancer ( 22 – 24 ), as well as lung repair ( 25 ), was also significantly enriched. Identifying secreted proteins upregulated in RA-treated HPMECs Lung repair requires coordinated crosstalk among alveolar cell types ( 26 ). Since RA promoted angiogenesis and wound healing in endothelial cells but had no direct effect on alveolar epithelial cells (Fig. 2 ), we hypothesised that RA signalling may induce the release of pro-repair signals from the endothelium to act in a paracrine manner on other alveolar cell populations. To identify candidate secreted factors, the 545 DEGs detected at 4 hours post RA-treatment were subjected to Panther “Protein classification” analysis. This revealed 14 candidate genes encoding intercellular signalling proteins, eight of which were upregulated, including CXCL8 (C-X-C motif chemokine ligand 8), TGFA (transforming growth factor alpha ) , BMP2 (bone morphogenetic protein 2), FGF20 (fibroblast growth factor 20), WNT9A (Wnt family member 9a), GDF15 (growth differentiation factor 15), EFNB1 (Ephrin B1), and ADM (adrenomedullin) (Table 1 ). Three genes, CXCL8, TGFA , and BMP2 , were selected for further analysis due to their known association with lung diseases and relevant biological functions ( 27 – 29 ). Notably, TGFα, encoded by TGFA , is a ligand for the EGFR signalling pathway, one of the top enriched pathways in RA-treated HPMECs (Fig. 4 B). Table 1 Identification of 14 genes encoding intercellular signalling proteins. Gene symbol Protein class Mapped to signalling pathway Fold Change FDR P -value CXCL8 chemokine CCKR signalling map, Interleukin signalling pathway, Inflammation mediated by chemokine and cytokine signalling pathway 9.24 2.56E-07 TGFA growth factor EGF receptor signalling pathway 4.56 0.0176 BMP2 growth factor TGF-beta signalling pathway, Gonadotropin-releasing hormone receptor pathway 3.57 0.0004 FGF20 growth factor FGF signalling pathway 2.88 0.0184 WNT9A intercellular signal molecule Wnt signalling pathway, cadherin signalling pathway 2.02 0.0376 GDF15 growth factor TGF-beta signalling pathway 1.79 0.0087 EFNB1 membrane-bound signalling molecule Angiogenesis 1.7 0.0286 ADM peptide hormone - 1.58 0.0303 CCL23 cytokine - -9.12 0.0188 WNT5A intercellular signal molecule Angiogenesis, Wnt signalling pathway, cadherin signalling pathway -8.18 0.0167 PDYN neuropeptide Enkephalin release, Opioid prodynorphin pathway, Opioid proenkephalin pathway -4.05 0.0241 PTN growth factor -3.34 0.0473 FGF5 growth factor FGF signalling pathway -2.22 0.0129 FGF8 growth factor FGF signalling pathway -2.11 0.0484 Interestingly, qRT-PCR validation confirmed the upregulation of BMP2 and CXCL8 in RA-treated HPMECs ( p < 0.05), consistent with the microarray data, but no significant changes were observed in TGFA transcript levels (Fig. 4 C). Additional top upregulated genes, HOXA3 and HOXA5 , were also validated by qRT-PCR, corroborating the microarray results (Figure S3 ). It is important to note that RNA for qRT-PCR validation was isolated from RA-treated HPMEC monolayers cultured on fibronectin-coated dishes, whereas RNA for the microarray analysis was obtained from RA-treated HPMECs undergoing angiogenesis. TGFα induces wound healing and cell migration in alveolar epithelial cells To evaluate the effects of TGFα, BMP2, and CXCL8 on wound healing and cell migration in alveolar epithelial cells, we treated A549 cells and primary human alveolar type 2 (hAT2) epithelial cells with recombinant proteins and performed scratch wound and cell migration assays, respectively. Treatment with TGFα significantly enhanced wound healing in A549 cells in a dose-dependent manner, with the maximal effect observed at 40 ng/ml ( p < 0.0001) compared to the untreated control (DMEM supplemented with 0.5% FBS). Treatment with 4 ng/ml TGFα also significantly increased wound closure ( p = 0.0149) (Figs. 5 A and B). Additionally, immunostaining with the proliferation marker Ki67, followed by quantification of Ki67-positive cells, revealed no significant differences in cell proliferation across the various concentrations of TGFα (Fig. 5 C). MTT assays further confirmed that TGFα did not affect cell viability at any concentrations tested. Cells treated with 70% methanol were used as a positive control for dead cells, which exhibited significant cell death compared to the untreated control ( p = 0.0003) (Figure S4 ). Next, we replicated the scratch assay in human primary alveolar type 2 (hAT2) cells isolated from resected lung tissue, following a previously described method ( 30 ). hAT2 cells exhibited low wound closure (10.5% to 20.8%) (Figure S5), likely due to the removal of collagen-I matrix in the scratch area, which impaired hAT2 migratory capacity. We then employed a cell migration assay to further assess the effect of TGFα on hAT2 cells. Based on the dosage-response observed in the scratch assay (Figure S5), we narrowed the range of TGFα concentrations for subsequent experiments to 0.2 ng/ml to 4 ng/ml. Consistent with findings in A549 cells, TGFα treatment enhanced hAT2 migration in a concentration-dependent manner, with the most pronounced effect observed at 4 ng/ml TGFα (42.5% cell migration rate) compared to 26.3% in the control ( p = 0.0314) (Figs. 5 D and E). TGFα treatment also did not significantly impact cell proliferation (Fig. 5 F), and labelling of hAT2 cells with NucView 488-caspase-3 dye indicated that TGFα did not induce apoptosis (Fig. 5 G). MTT assays confirmed that cell viability was maintained in TGFα-treated cells. Cells treated with 70% methanol served as a positive control for dead cells, demonstrating a significant reduction in cell viability compared to the untreated control (p < 0.0001) (Figure S6). The effects of BMP2 and CXCL8 on wound healing in A549 cells were also investigated; however, no significant differences were observed between treated and untreated cells at any concentrations tested (Figures S7A and B). MTT assays confirmed the concentrations of BMP2 and CXCL8 used in these experiments did not affect cell viability (Figures S7C and D). TGFα does not modulate cell migration or proliferation in human lung fibroblasts and microvascular endothelial cells To determine whether TGFα also influences cell migration in other alveolar cell populations, cell migration and scratch wound assays were performed on primary human lung fibroblasts (HLFs) and HPMECs, respectively. Our results demonstrated that TGFα did not significantly affect cell migration in HLFs (Figs. 6 A and B). Furthermore, immunostaining for the proliferation marker Ki67 revealed no effect of TGFα on cell proliferation. HLFs cultured in DMEM supplemented with 10% FBS exhibited a significantly higher percentage of Ki67-positive cells (42.9%) compared to control cells grown in DMEM containing reduced FBS (25.7%) ( p = 0.0172) (Fig. 6 C). As shown in Fig. 6 D, no apoptotic cells were detected in either control or TGFα-treated HLFs, whereas cells treated with 70% methanol served as a positive control (Fig. 6 D). Similarly, TGFα had no effect on wound healing in HPMECs (Figs. 6 E and F). Cell viability assays confirmed that TGFα treatment did not influence metabolic activities in either HLFs or HPMECs (Figures S8A and B). In summary, our findings demonstrate that the pro-migratory and wound healing effects of TGFα are specific to alveolar epithelial cells. Endothelial angiogenesis-induced TGFα modulates epithelial wound healing through activation of EGFR signalling Given that TGFα is a well-established ligand for EGFR signalling, we next investigated whether TGFα-induced epithelial wound healing is mediated through this pathway. First, to establish that EGFR activation could be reliably detected, A549 cells were treated with recombinant TGFα for 30 minutes or 2 hours and immunostained with an anti-phospho-EGFR antibody. As shown in Fig. 7 A, there was no noticeable difference in phospho-EGFR staining between the serum-free controls and TGFα-treated A549 cells after 30 minutes. Epidermal growth factor (EGF), another EGFR ligand, served as a positive control but similarly showed no change in EGFR staining at this early time point. However, after 2 hours, changes in the pattern of phospho-EGFR were observed in both TGFα- and EGF-treated cells, particularly at the cell membrane, which were not present in control cells (Figs. 7 B and C). This suggests that EGFR activation is detectable at 2 hours after TGFα stimulation. To determine whether TGFα secreted by endothelial cells during angiogenesis could activate EGFR in A549 cells, we employed a transwell co-culture system in which A549 cells were cultured in the upper chamber and HPMECs, treated with either RA or vehicle control (EtOH), were seeded on Matrigel in the lower chamber (Fig. 7 D). Remarkably, A549 cells co-cultured with RA-treated HPMECs showed marked changes in the localisation of phospho-EGFR staining compared to those co-cultured with EtOH-treated HPMECs (Figs. 7 E and F). These findings indicate that RA-induced angiogenesis in HPMECs activates EGF receptor in adjacent epithelial cells. To assess whether extracellular TGFα secreted from HPMECs contributed to this effect, we measured TGFα levels in conditioned media collected from EtOH- or RA-treated HPMECs at 4 and 16 hours post-treatment using ELISA. However, TGFα was undetectable in all samples. It should be noted that although the TGFα standard provided with the ELISA kit produced a reliable standard curve, media supplemented with the commercially available recombinant TGFα used in this study also yielded no detectable signal (Table S5). We were therefore unable to confirm the presence of secreted TGFα following RA treatment in this study. Discussion Here we conducted a microarray to identify secreted proteins that were upregulated in RA-treated HPMECs undergoing angiogenesis. The top three most upregulated of these proteins, CXCL8, TGFa and BMP2, were selected for further investigation. TGFa specifically induced repair and migration of both A549 and human primary AT2 cells, whereas CXCL2 and BMP2 did not. In contrast, TGFa had no effect on repair or proliferation of either primary HLFs or HPMECs. Subsequently, using a co-culture approach, we found that RA treatment induced HPMECs to undergo angiogenesis, which led to activation of the TGFa receptor EGFR, in adjacent A549 cells, indicative of a paracrine effect of RA. It has long been appreciated that RA is a powerful regenerative agent across organs and species ( 31 , 32 ). Interestingly, a recent study in the skin identified a pivotal role of RA in regulating stem cell fate during wound repair by modulating lineage plasticity; a transient state that bestows tissue stem cells with fate flexibility and is important in the repair response to injury ( 33 ). In the lungs, RA is critical for development, but it also has a powerful ability to regenerate lung alveoli ( 11 , 34 ). Epidemiological studies further demonstrate that Vitamin A deficiency has detrimental effects not only on the lungs but also other organ systems ( 35 ). Promising data from animal experiments and population studies led to clinical trials being conducted with the aim of determining whether RA might be an effective treatment for patients with emphysema. Although the results of these first human lung regenerative clinical trials were largely disappointing and failed to meet their primary end points, in one trial, involving patients with CT-confirmed smoking-related emphysema, post-hoc subgroup analysis found that patients with predominantly lower-zone disease experienced reduced declines in FEV1, gas diffusion, and exercise capacity compared with placebo ( 36 ). These findings highlight the need for deeper mechanistic understanding of the cellular and molecular mechanisms through which putative pro-repair factors such as RA exert their effects prior to clinical trials taking place. We previously showed that instead of directly inducing epithelial lung repair, RA induced angiogenesis of HPMECs ( 15 ) and we therefore hypothesised that RA might indirectly mediate alveolar epithelial repair in a paracrine manner by inducing secretion of a pro-repair signalling molecule(s) during angiogenesis. To investigate this, we combined the use of human and 3D ex vivo models with detailed mechanistic studies of RA driven lung repair. Our findings show that in support of our hypothesis, following RA-induced angiogenesis of HPMECs, TGFα is upregulated and this secreted growth factor specifically drives repair of human alveolar epithelial cells. TGFα is one of seven ligands that bind to EGFR with varying affinities ( 37 ). Considerable evidence has demonstrated a role for EGF ligands and EGFR signalling in lung repair ( 38 ). However, although TGFα is expressed in the lungs throughout the life course, its role in repair and regeneration remains unclear. Instead, adult lung studies have focused on the detrimental effects of aberrant TGFα expression. For example, overexpression of TGFα in mice leads to enlarged alveolar airspaces and fibrosis, although lower doses did not produce significant changes ( 17 ). Interestingly, TGFα is also upregulated in lung diseases characterised by tissue damage, including idiopathic pulmonary fibrosis (IPF), COPD, and in post-COVID patients ( 39 – 41 ). While this upregulation has often been interpreted as detrimental, it is equally plausible that increased expression reflects an adaptive repair response, with TGFα contributing positively to lung regeneration. Recently, Li and colleagues showed that lipopolysaccharide (LPS) injury induces upregulation of TGFα/EGFR signalling, leading to abnormal elastin deposition and alveolar simplification; inhibition of EGFR with erlotinib mitigated these effects on alveoli, underscoring the need for tight regulation of TGFα levels and highlighting the TGFα-EGFR axis as a potential therapeutic target to modulate alveolar regeneration ( 17 , 39 ). From our microarray data, we identified eight upregulated genes encoding secreted proteins, of which the top three were selected for detailed investigation (Table 1 ). Strikingly, only TGFα promoted epithelial repair. Furthermore, the pro-repair capacity of TGFα is specific to lung alveolar epithelial cells, as no effect was observed on the repair of human lung fibroblasts or HPMECs. Intriguingly, unlike the other candidate signalling molecules, TGFα mRNA was not upregulated in monolayers of RA-treated HPMECs but was increased in RA-treated HPMECs undergoing angiogenesis. This suggests that TGFA induction is not a direct consequence of RA treatment but rather a response stimulated during RA-induced angiogenesis, thereby supporting our hypothesis that a paracrine signal released following HPMEC angiogenesis drives epithelial repair. This contrasts with a previous study reporting suppression of TGFA expression in upper airway epithelial cells and airway explants treated with retinol ( 40 ). Notably, our study used retinoic acid (a derivative of retinol) at 10 µM compared with 0.1 µM retinol in Miller’s work and focused on alveolar cells rather than airway epithelial cells or tissues, suggesting that TGFA transcriptional responses to retinoids are tightly regulated, both dose- and context-dependently. The validity of the microarray data was confirmed by the demonstration that RA signalling was among the significantly enriched pathways in RA-treated HPMECs at 4 hr. GO enrichment analysis highlighted two main aspects of RA-induced changes: first, reorganisation of actin filaments, which governs cell adhesion and migration—processes essential for wound healing and tissue repair; and second, changes in epithelial cell behaviour, supporting the idea of crosstalk between HPMECs and epithelial cells in response to exogenous RA exposure. Although we identified a pro-repair effect of TGFα on epithelial cells and demonstrated EGFR activation in RA-treated A549 cells co-cultured with HPMECs undergoing angiogenesis, we were unable to detect TGFα in supernatants collected from these co-cultures at either 4 or 16 hours. The ELISA kit was validated with a standard curve, but no TGFα was detected in the co-culture supernatants, or even in culture media supplemented with recombinant TGFα, which was intended to serve as a positive control (Table S5). This may reflect methodological limitations, such as suboptimal timing of supernatant collection or insufficient recognition of specific TGFα isoform(s) by the antibodies used. Notably, Ensembl reports multiple isoforms of TGFα, which could contribute to antigen-antibody mismatch. Despite this limitation, we were able to show activation of EGFR signalling in A549 cells in response to RA-treated HPMECs undergoing angiogenesis in co-culture. Ideally, we would have further confirmed this by blocking a TGFα-specific EGFR phosphorylation site. However, no known TGFα-specific EGFR phosphorylation sites have been identified to date. Direct inhibition of TGFα could be considered, but this strategy risks confounding results, as the absence of TGFα may trigger compensatory activity from other EGFR ligands. Indeed, experimental evidence showed that loss of individual EGFR ligands, including TGFα, can be functionally compensated for by others, and only simultaneous inhibition of multiple ligands substantially reduces EGFR signalling ( 41 ). In summary, this study shows that RA induces angiogenesis in lung endothelial cells, leading to TGFα-dependent activation of EGFR, which drives repair of alveolar epithelial cells. More broadly, our findings highlight the importance of using physiologically relevant human models to uncover the detailed mechanisms of tissue repair required for the development of effective pharmacological regenerative medicine treatments. Methods Sex as a biological variable Human tissue, for cell isolation, was obtained from both males and females. Study approval Lung tissue samples were collected from male and female patients undergoing lung resection at the Royal Brompton & Harefield hospital (RBHT), by Biobank sample co-ordinators within the RBHT Clinical Research Facility (CRF). All samples were obtained following informed consent from patients. The research was conducted using the RBHT Biobank ethics number: 20/SC/0142. Mice All animal maintenance and procedures were conducted in compliance with the requirements of the Animal (Scientific Procedures) Act 1986. Animal work was approved by the South Kensington AWERB committee at Imperial College London. Mice were housed in specific pathogen-free conditions and given food and water ad libitum . Wild-type female mice aged 10–12 weeks were purchased from Charles River (United Kingdom). Mouse Precision-Cut Lung Slices (PCLS) and acid injury Mouse PCLS were generated following previously established protocols ( 42 , 43 ). Briefly, adult mice were euthanised via intraperitoneal injection of pentobarbital. The anterior chest and neck wall were carefully dissected, and a small incision was made in the trachea below the cricoid cartilage. A 21G rigid metal cannula was inserted into the trachea incision, and the lungs were inflated by injecting 1.2 ml of 2% (w/v) low-gelling agarose (Sigma–Aldrich, A9414) dissolved in 1× Hank’s Balanced Salt Solution (HBSS; Life Technology, 14025100) containing 1% HEPES buffer (Life Technology, 15630080). The chest cavity was cooled with ice to solidify the agarose. The lungs and heart were excised and submerged in ice-cold HBSS/HEPES buffer. Lung lobes were carefully separated and sliced transversely at a thickness of 300 µm using an automated vibratome (Compresstome VF-300-0Z; Precisionary Instruments LLC) in ice-cold HBSS/HEPES buffer. PCLS were then transferred to 24-well plates containing ice-cold serum-free (SF) Dulbecco’s Modified Eagle Medium (DMEM)-GlutaMAX (Life Technologies, 31966021) with 1% penicillin-streptomycin (Merck, P0781). PCLS were incubated overnight at 37°C in a 5% CO 2 atmosphere and washed three times with warm SF-DMEM to remove any remaining agarose. For acid injury, PCLS were treated with 0.1 M HCl (Fisher Chemical, 10053023) for 1 minute at room temperature (RT). The HCl solution was then discarded, and PCLS were washed three times with phosphate-buffered saline (PBS; Sigma; D8537) to remove residual acid. PCLS were cultured in fresh SF-DMEM for 48 hours before RNA extraction. Cell culture Primary human pulmonary microvascular endothelial cells (HPMECs; Promocell, C12281) and human umbilical vein endothelial cells (HUVECs; Promocell, C12200) were cultured in Endothelial Cell Growth Medium MV (EGM-MV; Promocell, C22020) and Endothelial Growth Media (EGM; Promocell, C-22010), respectively. Cells were maintained at 37°C in a 5% CO 2 atmosphere. Cells were used between passages 3 and 7, showing no changes in morphology or baseline tube-forming capacity. HPMECs used in this study were sourced from two Caucasian female donors, aged 64 and 98. A549 cells, a human alveolar adenocarcinoma epithelial cell line, were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM-GlutaMAX (Gibco) supplemented with 10% foetal bovine serum (FBS; Sigma, F9665) and 1% penicillin/streptomycin at 37°C with 5% CO 2 . Primary human alveolar type 2 (hAT2) epithelial cells and human lung fibroblasts (HLFs) were isolated from histologically normal lung tissues obtained from patients undergoing surgical resection for suspected lung tumours, through the Clinical Research Facility (CRF) Respiratory Biobank at Royal Brompton & Harefield Hospitals (Guy’s and St Thomas’ NHS Foundation Trust) (Ethics reference number: 20/SC/0142). The isolation protocol was based on a previously described method ( 30 ) with slight modifications. Briefly, lung tissue was perfused with HBSS (Invitrogen, 14025-050) to remove blood and debris, followed by digestion with 0.25% trypsin (Sigma, T8003) at 37°C for 45 minutes. Trypsinisation was halted by adding FBS, and the tissue was finely chopped. DNase I (Sigma, DN25) was added to facilitate degradation of extracellular DNA to improve tissue dissociation. The resulting tissue suspension was passed through a tea strainer and a 40-µm mesh filter (Marathon Lab Supplies, 352340) to remove undigested tissue. The filtrate was centrifuged at 1,300 rpm for 10 minutes at 12°C. The pellet was resuspended in hybridoma serum-free medium (SFM; Gibco, 11570416) containing 50 µg/ml DNase I and incubated at 37°C for 45 minutes to remove immune cells. After incubation, the supernatant was collected and centrifuged again at 1,300 rpm for 10 minutes at 12°C. The pellet was resuspended in hybridoma medium containing 10% FBS and incubated at 37°C for 2 hours. Cells adhering to the dishes after this period were identified as HLFs, which were subsequently cultured in DMEM-GlutaMAX supplemented with 10% FBS for downstream experiments. The supernatant containing non-adherent cells was centrifuged at 1,300 rpm for 10 minutes at 12°C, and the resulting pellet, containing hAT2 cells, was resuspended and cultured in hybridoma medium with 10% FBS. hAT2 cells were not passaged, and all experiments involving these cells were completed within 68 hours of seeding. HLFs were used between passages 3–7 for experiments. The specificity of isolated hAT2 cell populations was confirmed by immunostaining with alveolar cell type-specific markers. The hAT2 cells were positive for pan-cytokeratin (a pan-epithelial marker) and Pro-SPC (an AT2 cell marker), but negative for vimentin (a fibroblast marker) and podoplanin (PDPN; an AT1 cell marker) (Figure S9). Pharmacological treatment Retinoic acid (RA; Sigma, R2625) used in this study was dissolved in absolute ethanol (EtOH; Sigma-Aldrich, 51976). Recombinant human TGFα (catalogue number: 239-A1-100), BMP2 (catalogue number: 355-BM-010), and CXCL8 (catalogue number: 208-IL-010) were obtained from R&D Systems and reconstituted according to the manufacturer’s instructions. Human EGF was sourced from Promocell (catalogue number: C-39220). Matrigel-based angiogenesis assay and tube quantification Angiogenesis µ-slides (Ibidi, 81506) or 24-well plates (Ibidi, 2401) were pre-coated with growth factor-reduced Matrigel (BD Biosciences, 354230), diluted to a concentration of 5 mg/ml with pre-chilled endothelial cell basal medium (EBM; Promocell, C-22220) without supplements. The plates were incubated at 37°C for 1 hour to allow the Matrigel to solidify. HPMECs were serum-starved for 24 hours prior to conducting the tube formation assays. Cells were seeded at a density of 4,750 cells per well for the angiogenesis µ-slides and 70,000 cells per well for the 24-well plates. Cells were treated with either the vehicle (EtOH) or 10 µM RA. For time-lapse imaging of tube formation, the µ-slide was immediately transferred to a pre-equilibrated, humidified incubator chamber (37°C, 5% CO 2 , and approximately 21% oxygen levels) of an inverted Zeiss Axio Observer widefield epifluorescence microscope. Imaging was performed over a 4-hour period using an EC Plan-Neofluar 10×/0.30 objective lens. The total tube length and the total number of nodes per field of view were quantified using FIJI software. Data were expressed as the mean total tube length or total number of nodes per field of view. RNA extraction and quantitative real-time PCR (qRT-PCR) Mouse PCLS were homogenised using the FastPrep-24TM Tissue Homogeniser (MP Biomedicals), and total RNA was extracted with the RNeasy mini kit (Qiagen, 74104) following the manufacturer’s protocols. For each experimental condition, RNA samples were pooled from three PCLS, and a total of three RNA samples were prepared per condition. Additionally, RNA was extracted from HPMEC monolayers using the same RNeasy mini kit. RNA concentration and quality were assessed using the TapeStation 2200 (Agilent), and only samples with an RNA integrity number (RIN) greater than 8 were used for downstream cDNA synthesis and qRT-PCR. Approximately 200 ng of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to manufacturer’s instructions. Quantitative RT-PCR was then performed using TaqMan Fast Advanced Master Mix (Life Technologies) on a 7500 Fast Real-Time PCR system (Applied Biosystems). The housekeeping gene B2m was used as a reference for normalisation. Relative gene expression levels were calculated using the 2 − ΔΔCT method. Each sample was tested in triplicate, with four biological replicates derived from four different mice. The following TaqMan primers (Life Technologies) were used in this study: mouse B2m (Mm00437762_m1), human B2M (Hs00984230_m1), Rara (Mm01296312_m1), Rarb (Mm01319677_m1), Rarg (Mm00441083_m1), Aldh1a1 (Mm00657317_m1), Aldh1a2 (Mm00501306_m1), Aldh1a3 (Mm00474049_m1), Cyp26a1 (Mm00514486_m1), Cyp26b1 (Mm00558507_m1), Cyp26c1 (Mm03412454_m1), TGFA (Hs00608187_m1), CXCL8 (Hs00174103_m1), BMP2 (Hs00154192_m1), HOXA3 (Hs00601076_m1), and HOXA5 (Hs00430330_m1). Microarrays and raw data processing Microarray service was provided by UK Bioinformatics Ltd. (UKB, UK). Total RNA was extracted from HPMECs cultured on Matrigel using Trizol reagent (Invitrogen, UK). RNA quality was assessed using the Agilent 2100 Bioanalyzer. For each treatment and time point, four RNA samples were prepared from independent experiments, with each sample pooled from three technical replicates (see Table S6 for sample details). cDNA was synthesised using the Ovation® Pico WTA System V2 and labelled with the Encore Biotin Module, following the manufacturer’s protocols (Nugen, USA). The labelled cDNA was hybridised to Affymetrix Human U133 Plus 2.0 Gene Chips (Affymetrix) according to the manufacturer’s instructions. The resulting microarray data were generated in an Affymetrix CEL format and processed using the Transcriptome Analysis Console (TAC) software v4.0.2 (Thermo Scientific). Raw data normalisation and summarisation were performed using the MAS5 algorithm within the TAC software. Gene expression analysis was conducted using the human genome reference assembly hg19. Differential expression was defined by a fold change threshold of > 1.5 or < -1.5, and a false discovery rate (FDR)-adjusted p -value of < 0.05. Statistical significance was calculated using moderated t-statistics from the empirical Bayes (ebayes) method within an ANOVA framework, allowing for robust identification of differentially expressed genes between RA-treated and EtOH-treated control samples. FDR correction was applied using the Benjamini-Hochberg algorithm to control for multiple testing. Gene Set Enrichment Analysis (GSEA) GSEA was performed to identify pathways and biological processes significantly associated with upregulated genes. Pathway enrichment analysis was conducted using PANTHER version 19.0 ( www.pantherdb.org/ ), focusing on PANTHER Pathways and Reactome Pathways. For the enrichment of Gene Ontology (GO) biological processes, Cytoscape software version 3.10.2 was utilised, in conjunction with the STRING enrichment tool. Enriched pathways and biological processes were ranked according to their statistical significance. Scratch wound and cell migration assays For the scratch wound assay, 45,000 HUVECs, HPMECs, or A549 cells were seeded onto 8-well Permanox chamber slides (Fisher Scientific; 16250681), pre-coated with 2 µg/ml fibronectin (Sigma, F1141) for HUVECs and HPMECs, or 50 µg/ml rat-tail collagen I (Gibco, A1048301) for A549 cells. Cells were cultured in their respective complete media - EGM for HUVECs, EGM-MV for HPMECs, and DMEM-GlutaMAX supplemented with 10% FBS for A549, and incubated overnight at 37°C with 5% CO 2 to reach confluence. A scratch wound was made across the centre of the confluent monolayer using a P1000 pipette tip. Wells were gently rinsed with PBS to remove dislodged cells, followed by the addition of appropriate treatments. For RA treatments, HUVECs were exposed to 3 µM, 5 µM, 10 µM, 20 µM, or 50 µM RA, diluted in EBM. A549 cells were treated with 10 µM of RA in SF-DMEM. Since RA was reconstituted in EtOH, control media containing 0.1% EtOH in either EBM or SF-DMEM were used for HUVECs and A549 cells, respectively. Positive control media for enhanced wound healing consisted of EGM with 3% FCS for HUVECs and DMEM supplemented with 10% FBS for A549 cells. For the cell migration assay, hAT2 and HLFs were plated using 2-well silicone culture inserts (Ibidi, 80209) adhered to 50 µg/ml rat-tail collagen I-coated 13 mm coverslips placed in 24-well plates. Freshly isolated hAT2 cells were seeded at a density of 250,000 cells per well, while HLFs were plated at 15,000 cells per well. For HLFs, the silicone inserts were removed after overnight incubation. For hAT2 cells, debris and dead cells were gently washed off with PBS 24 hours post-seeding, and fresh hybridoma SFM supplemented with 10% FBS was added, followed by a further 24-hour incubation before insert removal. TGFα treatments were administered at appropriate concentrations in DMEM with 1% FBS for HLFs or in hybridoma SFM with 1% FBS for hAT2 cells. Cells in both assays were imaged at time points t = 0 h and the endpoint at t = 19 h or 24 h using an inverted Zeiss Axio Observer widefield epifluorescence microscope housed in a pre-equilibrated, humidified incubator chamber (37°C, 5% CO 2 , ~ 21% oxygen). Images were captured using an EC Plan-Neofluar 10×/0.30 objective lens, and tiling was employed to image the cell-covered areas. Wound healing percentage was quantified using a FIJI macro developed by Steve Rothery (Facility for Imaging by Light Microscopy, Imperial College London) or through manual analysis. For manual quantification, the wound areas were outlined with the freehand tool in FIJI, and the percentage healed was calculated by subtracting the endpoint area from the area at t = 0 h. Three independent experiments were conducted, and each experiment was run in triplicate. Immunofluorescence and imaging Cells were fixed in 4% paraformaldehyde (PFA) for 10 minutes at RT, followed by PBS. Permeabilisation was carried out using 0.2% (v/v) Triton X-100 in PBS RT for 5 minutes at RT. Blocking was then performed for 1 hour at RT using PBSBT buffer, which contains 3% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS. After blocking, cells were incubated overnight at 4°C with primary antibodies diluted in PBSBT blocking buffer. The following primary antibodies were used in this study: rabbit anti-vimentin (Bioorbyt, orb304659; 1:200), mouse anti-pan-cytokeratin (Sigma–Aldrich, C2931; 1:200), rabbit anti-Pro-SP-C (Abcam, ab90716; 1:300), hamster anti-PDPN (Developmental Studies Hybridoma Bank, 8.1.1; 1:50), rat anti-Ki67 (eBioscience, 14-5698-82; 1:500), and rabbit anti-phospho-EGFR Y1068 (Cell Signalling Technology, 3777S; 1:100). Following three washes with PBS, cells were incubated for 1 hour at RT with rhodamine phalloidin (Biotium, 00027; 1:300) and species-specific Alexa Fluor 488 and 647 secondary antibodies (Thermo Fisher Scientific; 1:500). After further PBS washes, cell nuclei were stained with DAPI (Thermo Scientific, 62249). Coverslips were mounted with ProLong Gold Antifade Mountant (Invitrogen, P36930). Imaging was performed using a Leica SP8 inverted confocal microscope equipped with a HC PL APO 10×/0.40 air objective lens or a HC PL APO 40×/1.30 oil objective lens. For some experiments, channel colours were changed during image post-processing to improve visual clarity. MTT assay To evaluate the impact of pharmacological treatments on cell viability, an MTT assay was conducted according to the manufacturer’s instructions (Roche, 11465007001). Briefly, 150 µl of 10% MTT solution diluted in serum-free media was added to each well of a 96-well plate and incubated at 37°C for 45 minutes. The resulting formazan crystals, which formed in viable cells, were solubilised by adding an equal volume of DMSO and incubating at 37°C for 10 minutes. 100 µl of eluted formazan solution was measured spectrophotometrically, with absorbance recorded at 570 nm and corrected at 690 nm using a plate reader. Caspase-3 assay To identify apoptotic cells, the NucView® 488 Caspase-3 assay kit (Biotium; 30029-T) was employed, following the manufacturer’s protocols. At the end point of pharmacological treatments, cells were incubated with 5 µM NucView 488 substrate (prepared as 1:40 stock dilution) in the dark at RT for 30 minutes. After incubation, the dye was removed, and cells were gently washed three times with PBS. Cells were then fixed with 4% PFA for 10 minutes at RT. After fixation, the PFA was discarded, and the cells were washed three times with PBS. Cells were counterstained with DAPI at RT for 5 minutes and washed once with PBS. Cells were then mounted using ProLong® Gold Antifade Reagent and imaged on the same day using a Leica SP8 inverted confocal microscope equipped with a HC PL APO 40×/1.30 oil objective lens. Transwell insert-based co-culturing of HPMECs and A549 HPMECs and A549 cells were co-cultured as illustrated in Fig. 7 D. A549 cells were plated at a density of 37,000 per well in DMEM supplemented with 10% FBS onto a 24-well transwell insert with 0.4-µm pore polyethylene terephthalate (PET) membrane (Brand, BR782711) pre-coated with 50 µg/ml rat tail collagen-I. The cells were incubated overnight at 37°C in a 5% CO 2 atmosphere. The following day, HPMECs were seeded on Matrigel and maintained in EBM as detailed in the methods section on “Matrigel-based angiogenesis assay and tube quantification”. HPMECs in the lower chamber were treated with 0.1% EtOH or 10 µM RA. Next, the DMEM from the transwell inserts containing A549 cells was discarded, and the cells were washed three times with PBS to remove any residual FBS. The media in the inserts was then replaced with EBM supplemented with 0.2% FCS. The transwell inserts containing A549 cells were subsequently placed onto the HPMECs, allowing for co-culture durations of 2 hours, 4 hours, or 16 hours. After 2 hours of co-culture, A549 cells in the upper chamber were fixed and immunostained with phospho-EGFR, while culture supernatants from the HPMEC were collected at the 4-hour and 16-hour time points to assess extracellular TGFα levels using the Human TGF-alpha DuoSet ELISA (R&D Systems, DY239) following the manufacturers’ instructions. Statistical analysis All graphs and statistical tests were produced using GraphPad Prism 10. Data are expressed as mean ± standard error of mean (SEM). The D’Agostino–Pearson test was employed to assess the normality of the data. For non-parametric datasets, the Mann–Whitney U -test was utilised for comparisons between two groups, while comparisons involving more than two groups were performed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. The specific statistical tests applied, sample size ( n ), and the number of experiments conducted are detailed in the figure legends. A p- value of less than 0.05 was considered statistically significant. Declarations Competing interests: All authors declare no financial or non-financial competing interests. Funding This study was funded by the Royal Brompton and Harefield Hospitals Charity (Grant 123/P90719) and an award from Mr and Mrs Youssef Mansour. Imaging for this study was conducted in the Facility for Imaging by Light Microscopy (FILM) at Imperial College and was part supported by Wellcome Trust (grant 104931/Z/14/Z). Funders played no role in study design, data collection, analysis, data interpretation of data, or the writing of this manuscript. Author Contribution CD, MH and S-SC conceived and designed the study. S-SC, CY, RM and DC performed experiments and analysed data. DC and MG provided conceptual advice. S-SC and CD wrote the manuscript. All authors contributed to editing the manuscript. MH and CD acquired funding. 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1","display":"","copyAsset":false,"role":"figure","size":123893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased RA signalling activity following acid injury in mouse PCLS. \u003c/strong\u003eHistogram showing transcript levels of genes associated with the RA signalling pathway, including RA receptors (\u003cem\u003eRara, Rarb\u003c/em\u003e, and \u003cem\u003eRarg\u003c/em\u003e), RA synthesis genes (\u003cem\u003eAldh1a1, Aldh1a2\u003c/em\u003e, and \u003cem\u003eAldh1a3\u003c/em\u003e), and RA degradation genes (\u003cem\u003eCyp26a1\u003c/em\u003e, \u003cem\u003eCyp26b1\u003c/em\u003e, and \u003cem\u003eCyp26c1\u003c/em\u003e), assessed 48 hours after injuring mouse PCLS with 0.1 M HCl. Gene expression was normalised to the housekeeping gene \u003cem\u003eB2m \u003c/em\u003eand expressed as fold change relative to uninjured control PCLS. \u003cem\u003en = \u003c/em\u003e4 mice; Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/788bc3af350ca1bc3c4f798e.png"},{"id":96708775,"identity":"fb8c7f56-fd12-47c3-9e8e-24a543174dd4","added_by":"auto","created_at":"2025-11-25 10:05:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":390664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRA enhances wound healing and angiogenesis in endothelial cells but does not affect alveolar epithelial A549 cells. (A-B) \u003c/strong\u003eRepresentative images showing endothelial tube-like structures. Tube lengths are depicted as green lines, and nodes are indicated by red dots. \u003cstrong\u003e(C-D)\u003c/strong\u003e Quantification of total tube length\u003cstrong\u003e (C) \u003c/strong\u003eand number of nodes\u003cstrong\u003e (D)\u003c/strong\u003e per field of view in HPMECs treated with either EtOH control or 10 μM RA at 90 minutes post-treatment. \u003cem\u003en\u003c/em\u003e = 5 independent experiments; each experiment was run in triplicate; each dot represents the mean value per experiment. Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05. \u003cstrong\u003e(E) \u003c/strong\u003eRepresentative images of HUVEC wound closure following treatment with 3 μM, 5 μM, and 10 μM RA, compared to EGM positive control and EBM + 0.1% EtOH vehicle controls, at 0 and 19 hours post-scratch. Wound edges are marked with yellow lines.\u003cstrong\u003e (F) \u003c/strong\u003ePercentage of wound healed measured at 19 hours post-scratch in HUVECs. \u003cem\u003en\u003c/em\u003e = 3 independent experiments; two or three technical replicates per experiment; each dot represents a technical replicate. Data are presented as mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(G) \u003c/strong\u003eImages of HUVECs treated with 20\u003cstrong\u003e \u003c/strong\u003eμM and 50 μM RA at 0 and 19 hours post-scratch. \u003cstrong\u003e(H) \u003c/strong\u003eHistogram showing the percentage of wound healing in A549 cells treated with 10 μM RA compared to DMEM + 10% FBS positive control and serum-free DMEM + 0.1% EtOH vehicle controls at 0 and 19 hours post-scratch.\u003cem\u003e n\u003c/em\u003e = 3 independent experiments; each dot represents the mean percentage of wound healed per experiment. Data are presented as mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/cb7f20159b19e9ca704b88b5.png"},{"id":96708650,"identity":"5565fa63-3094-4412-affa-2cc2300fd141","added_by":"auto","created_at":"2025-11-25 10:04:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":220676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPMECs respond to exogenous RA at 4 hours post-treatment. (A) \u003c/strong\u003eSchematic diagram of the HPMEC angiogenesis assay and the timeline for RNA extraction for microarray profiling using Affymetrix Human U133 Plus 2 GeneChip microarrays. Total RNA was extracted from HPMECs seeded on Matrigel at 0 hours, 40 minutes, and\u003cem\u003e \u003c/em\u003e4 hours after treatment. \u003cem\u003en\u003c/em\u003e = 4 independent experiments; each RNA sample was produced by pooling three technical replicates. \u003cstrong\u003e(B) \u003c/strong\u003eVolcano plot of differentially expressed genes (DEGs) in HPMECs treated with 10 μM RA versus EtOH control at 4 hours. Upregulated genes are shown in red, downregulated genes in green. \u003cstrong\u003e(C) \u003c/strong\u003eExtrapolated volcano plot highlighting upregulation of key RA signalling components, including \u003cem\u003eRARB, DHRS3\u003c/em\u003e, \u003cem\u003eNRIP1\u003c/em\u003e, and \u003cem\u003eRDH10\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eat 4 hours after RA treatment.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/5cde3aea6a155aa42abee74d.png"},{"id":96621928,"identity":"9c95c822-d5fb-4506-a327-9621f7ac3fb9","added_by":"auto","created_at":"2025-11-24 11:05:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene set enrichment analysis and validation of expression change in secreted protein-encoding genes. (A) \u003c/strong\u003eHistogram showing Gene Ontology (GO) biological process terms enriched in upregulated genes in RA-treated HPMECs, analysed using the Cytoscape STRING enrichment tool. \u003cstrong\u003e(B) \u003c/strong\u003eOver-represented pathways in RA-treated HPMECs identified by\u003cstrong\u003e \u003c/strong\u003ePANTHER and Reactome pathway analyses.\u003cstrong\u003e \u003c/strong\u003eBiological processes and pathways are ranked by statistical significance. \u003cstrong\u003e(C) \u003c/strong\u003eTranscript levels of selected secreted protein genes, including \u003cem\u003eTGFA, BMP2\u003c/em\u003e, and \u003cem\u003eCXCL8\u003c/em\u003e, in HPMECs treated with EtOH control or 10 μM RA. HPMEC monolayers were cultured on fibronectin-coated plates before treatment. \u003cem\u003en\u003c/em\u003e = 4 independent experiments; each experiment was run in triplicate. Data are presented as mean ± SEM; Mann–Whitney \u003cem\u003eU-\u003c/em\u003etest, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/02ff6546c9e6f1ca43971710.png"},{"id":96708410,"identity":"5d68a530-6509-4120-b9b7-9d66ff392422","added_by":"auto","created_at":"2025-11-25 10:01:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":348242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGFα promotes wound healing and cell migration in A549 and primary human AT2 cells. (A) \u003c/strong\u003eRepresentative images showing A549 cells treated with DMEM + 10% FBS (positive control), DMEM + 0.5% FBS (negative control), or 40 ng/ml TGFα at 0 and 24 hours post-scratch. Wound edges are marked with green lines. \u003cstrong\u003e(B)\u003c/strong\u003eHistogram of wound closure at 24 hours post-scratch in A549 cells treated with controls or increasing concentrations of TGFα (0.1 ng/ml, 0.2 ng/ml, 0.4 ng/ml, 4 ng/ml, and 40 ng/ml). \u003cstrong\u003e(C) \u003c/strong\u003ePercentage of Ki67-positive cells in A549 treated with varying TGFα concentrations, normalised to total DAPI-positive nuclei. \u003cem\u003en\u003c/em\u003e = 3 independent experiments; each experiment was run in duplicate or triplicate. Data are presented as mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(D)\u003c/strong\u003e Representative images of hAT2 cells treated with hybridoma medium + 1% FBS (negative control) or 4 ng/ml TGFα at 0 and 24 hours post-migration. Green lines indicate cell-free areas. \u003cstrong\u003e(E)\u003c/strong\u003eQuantification of hAT2 migration at 24 hours after treatment with hybridoma medium + 10% FBS (positive control), hybridoma medium + 1% FBS (negative control), or increasing TGFα concentrations (0.2 ng/ml, 0.4 ng/ml, 1 ng/ml, and 4 ng/ml). \u003cstrong\u003e(F) \u003c/strong\u003eQuantification of\u003cstrong\u003e \u003c/strong\u003eKi67-positive hAT2 cells treated with different TGFα concentrations. \u003cem\u003en\u003c/em\u003e = hAT2 cells isolated from 3 donors; two or three technical replicates per experiment; each dot represents a technical replicate. Data are presented as mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test, *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(G) \u003c/strong\u003eImages showing hAT2 cells labelled with NucView® 488 Caspase-3 dye (green) to detect apoptotic cells at the experiment endpoint. Nuclei were counterstained with DAPI (blue). Hybridoma medium + 1% FBS served as negative control; 70% methanol as positive control for apoptosis. Images were captured using an inverted Zeiss Axio Observer widefield epifluorescence microscope.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/123f506ee2eec30449b6164c.png"},{"id":96621932,"identity":"7a0fdf65-4bc1-474e-a377-e8220a518b59","added_by":"auto","created_at":"2025-11-24 11:05:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":306628,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGFα does not affect cell migration in HLFs or HPMECs. (A) \u003c/strong\u003eRepresentative images of HLFs treated with DMEM + 1% FBS (negative control) or 4 ng/ml TGFα at 0 and 16 hours post-scratch. White lines indicate wound areas. \u003cstrong\u003e(B)\u003c/strong\u003e Histogram illustrating cell migration rates at 16 hours in HLFs incubated with DMEM + 10% FBS (positive control), DMEM + 1% FBS (negative control), and varying concentrations of recombinant TGFα (0.2 ng/ml, 0.4 ng/ml, and 4 ng/ml). \u003cstrong\u003e(C) \u003c/strong\u003ePercentage of Ki67-positive HLFs after treatment with different concentrations of TGFα. \u003cem\u003en\u003c/em\u003e = HLFs isolated from 3 donors; two or three technical replicates per experiment; each dot represents a technical replicate. Data are presented as mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003e(D)\u003c/strong\u003eRepresentative images of HLFs stained with NucView® 488 Caspase-3 dye (green) and DAPI (cyan). DMEM + 1% FBS served as negative control; 70% methanol as positive control. Images were captured on a Leica SP8 inverted confocal microscope with a HC PL APO 40×/1.30 oil objective lens. \u003cstrong\u003e(E-F)\u003c/strong\u003e Wound closure at 0 and 16 hours \u003cstrong\u003e(E), \u003c/strong\u003eand quantification of wound healing \u003cstrong\u003e(F)\u003c/strong\u003ein HPMECs treated with EBM + 5% FCS (positive control), EBM + 2% FCS (negative control), 4 ng/ml, or 40 ng/ml TGFα. Green lines indicate wound edges. \u003cem\u003en\u003c/em\u003e = 1 experiment; two or three technical replicates per experiment; each dot represents a technical replicate. Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/384e25b4769cd4b80dbc8656.png"},{"id":96621934,"identity":"e135a743-0014-4057-adc5-602f7db7c81a","added_by":"auto","created_at":"2025-11-24 11:05:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":497432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGFα secreted during endothelial angiogenesis stimulates epithelial wound healing via EGFR activation. (A-C) \u003c/strong\u003eRepresentative images of A549 cells untreated (serum-free medium, negative control) or treated with 40 ng/ml TGFα or 50 ng/ml EGF for 30 minutes \u003cstrong\u003e(A)\u003c/strong\u003e or 2 hours \u003cstrong\u003e(B-C)\u003c/strong\u003e. Cells were immunostained for phospho-EGFR Y1068 (magenta), and nuclei were counterstained with DAPI (cyan). Images were captured using a Leica SP8 inverted confocal microscope with a HC PL APO 40×/1.30 oil objective lens. \u003cem\u003en\u003c/em\u003e = 2 independent experiments; three technical replicates per experiment. \u003cstrong\u003e(D) \u003c/strong\u003eSchematic of\u003cstrong\u003e \u003c/strong\u003ethe HPMEC–A549 transwell co-culture system.\u003cstrong\u003e \u003c/strong\u003eA549 cells were seeded on rat tail collagen I-coated 0.4 μm PET membrane inserts; HPMECs were seeded on Matrigel in the lower chamber. EtOH or RA was added to HPMECs, and co-cultured for 2, 4, or 16 hours. \u003cstrong\u003e(E-F)\u003c/strong\u003e Representative images showing A549 cells co-cultured for 2 hours with EtOH control-treated \u003cstrong\u003e(E)\u003c/strong\u003e or RA-treated \u003cstrong\u003e(F)\u003c/strong\u003e HPMECs. A549 cells were immunostained for phospho-EGFR (magenta) and nuclei counterstained with DAPI (cyan). Images were captured on a Leica SP8 inverted confocal microscope with a HC PL APO 40×/1.30 oil objective lens.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/93dc43a91cbf89104dec8917.png"},{"id":97135859,"identity":"576a3278-6577-454d-b8e1-0814679721a7","added_by":"auto","created_at":"2025-12-01 09:54:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3177051,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/f8eb1caf-fdec-4dee-8ed0-145324277998.pdf"},{"id":96708612,"identity":"c690f2fe-05a4-4472-b9af-79d895976d4d","added_by":"auto","created_at":"2025-11-25 10:04:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3425968,"visible":true,"origin":"","legend":"","description":"","filename":"CheongetalSupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/dd7641fa0594524cb4232187.pdf"},{"id":96709104,"identity":"baaa141c-6a80-43e1-aa8c-6f061f0bc5df","added_by":"auto","created_at":"2025-11-25 10:07:41","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":131001,"visible":true,"origin":"","legend":"","description":"","filename":"CheongetalSupplementaryData.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/c520c920f90d492148c74bf6.xlsx"},{"id":96708697,"identity":"d2858821-2029-46e9-83a4-016ed613d296","added_by":"auto","created_at":"2025-11-25 10:05:07","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":689006,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS110uMRA.avi","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/ab07f22b1fd3ed824d72350d.avi"},{"id":96621931,"identity":"7ee9bc84-4fa1-4be0-95ec-7dc3611b9901","added_by":"auto","created_at":"2025-11-24 11:05:37","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":705534,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS2EtOH.avi","url":"https://assets-eu.researchsquare.com/files/rs-7704784/v1/2e0a22982388324651c97786.avi"}],"financialInterests":"No competing interests reported.","formattedTitle":"TGF-alpha/EGFR signalling mediates retinoic acid-induced lung repair","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA common denominator in many lung diseases is insufficient functional alveolar surface area for gas-exchange. The root cause of this may differ, for example in emphysema, a pathology associated with chronic obstructive pulmonary disease (COPD), the alveoli are destroyed due to repeated insults to the lungs causing tissue destruction. In the case of bronchopulmonary dysplasia, a disease of prematurity, infants fail to form an appropriate number of alveoli, leading to compromised gas-exchange requiring oxygen support and life-long increased susceptibility to other lung diseases. Currently there are no treatments capable of restoring damaged lung parenchyma but one promising avenue through which this might be achieved, is to develop a regenerative medicine treatment to repair or (re)grow alveoli (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe lungs possess significant ability to regenerate and repair following injury, and we are beginning to understand the underlying cellular mechanisms governing these processes (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). For example, there are populations of tissue stem or progenitor cells that reside at different anatomical locations in the lungs which can become activated upon lung injury and contribute to regenerating the tissue (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In addition, other cell behaviours such as cell migration, proliferation, and cell plasticity contribute to repair of lung tissue (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Pharmacological-based modification of alveolar repair has begun to show real therapeutic potential for treating parenchymal lung diseases (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOne possible approach to develop a regenerative medicine treatment is to identify the molecular signals that drive endogenous lung repair/regeneration and harness these to repair damaged lung tissue. However, this requires detailed knowledge about the endogenous pro-repair pathways. Emerging lung pro-repair factors include Wingless-type MMTV integration site family (Wnts), fibroblast growth factors (FGFs), and sonic hedgehog growth factors (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In addition, the Vitamin A metabolite, retinoic acid (RA) plays a critical role in lung development and alveolar regeneration (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo date, research on alveolar repair and regeneration has largely focused on the epithelial population, with comparatively less attention given to the capillary endothelium, despite its equally critical role in gas exchange (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). RA is a well-known pro-regenerative factor across species, and studies in animal models demonstrated its ability to drive alveolar regeneration (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Human studies identified an imbalance between synthesis and breakdown of RA metabolism in emphysematous lungs, and RA signalling was activated in the microvascular endothelium, which stimulates angiogenesis but does not directly induce epithelial repair (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In addition, Ding and colleagues showed that endothelial-derived angiocrine signals induce and sustain alveolar regeneration (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Capitalising on these research findings, we hypothesised that factors secreted from the microvascular endothelium during RA-induced angiogenesis act as endogenous pro-repair mediators that drive alveolar epithelial repair in a paracrine manner. To test this hypothesis, we conducted microarray analysis of RA-treated lung microvascular endothelial cells undergoing angiogenesis and identified several candidate secreted proteins. We subsequently tested the pro-repair capabilities of these secreted proteins in vitro and found that transforming growth factor-α (TGFα), secreted from the RA-activated microvascular endothelium, promotes repair of the human alveolar epithelium via epidermal growth factor receptor (EGFR). TGFα, one of seven known EGFR ligands, has previously been shown to be important for alveologenesis (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These key mechanistic findings advance our understanding of how RA mediates lung repair and provide a foundation for developing evidence-based regenerative strategies to treat chronic lung diseases.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIncreased RA signalling activity following acid injury in mouse Precision-Cut Lung Slices (PCLS)\u003c/h2\u003e\u003cp\u003eTo explore the role of RA signalling pathway in lung repair, we generated mouse PCLS and conducted quantitative real-time PCR (qRT-PCR) to assess the expression of RA pathway components in uninjured controls versus acid-injured PCLS. Acid injury was induced by exposure to 0.1 M HCl for 1 min, and mRNA levels of RA receptors (\u003cem\u003eRara, Rarb\u003c/em\u003e, and \u003cem\u003eRarg\u003c/em\u003e), RA synthesis genes (\u003cem\u003eAldh1a1, Aldh1a2\u003c/em\u003e, and \u003cem\u003eAldh1a3\u003c/em\u003e), and RA degradation genes (\u003cem\u003eCyp26a1\u003c/em\u003e, \u003cem\u003eCyp26b1\u003c/em\u003e, and \u003cem\u003eCyp26c1\u003c/em\u003e) were quantified 48 hr post-injury. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cem\u003eRarb\u003c/em\u003e and \u003cem\u003eRarg\u003c/em\u003e transcript levels were significantly increased by 4.9-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029) and 2.3-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029), respectively, following acid injury of mouse PCLS. The RA synthesis gene \u003cem\u003eAldh1a3\u003c/em\u003e was also significantly upregulated, showing a 2.2-fold increase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029), whereas \u003cem\u003eAldh1a2\u003c/em\u003e expression decreased to 0.6-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029). Additionally, the RA degradation genes \u003cem\u003eCyp26a1\u003c/em\u003e and \u003cem\u003eCyp26c1\u003c/em\u003e showed significant upregulation with increases of 2.5-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029) and 1.6-fold (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029), respectively, compared to uninjured control PCLS. No significant changes were observed for \u003cem\u003eRara\u003c/em\u003e, \u003cem\u003eAldh1a1\u003c/em\u003e, or \u003cem\u003eCyp26b1\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCollectively, these data suggest an increased RA signalling activity following acid injury, highlighting a role for RA signalling in the early stages of lung repair.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRA stimulates angiogenesis and wound healing in endothelial cells, but not in alveolar epithelial cells\u003c/h3\u003e\n\u003cp\u003eOur previous work demonstrated that RA induced angiogenesis in human pulmonary microvascular endothelial cells (HPMECs) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). As a prelude to microarray experiments, we replicated the experiment. Consistent with previous findings, we found that tube length, averaging 40,546 \u0026micro;m per field of view with RA treatment, compared to 32,225 \u0026micro;m in the 0.1% vehicle ethanol (EtOH) control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0317) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Furthermore, the total number of nodes per field of view increased from 141 in the control to 217 in RA-treated HPMECs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0317) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess whether RA also modulates wound healing capacity in other endothelial cell types, scratch wound assays were performed on human umbilical vein endothelial cells (HUVECs) treated with varying concentrations of RA. Compared to vehicle EtOH-treated controls, RA significantly enhanced wound healing in a dose-dependent manner. The most pronounced effect was observed at 10 \u0026micro;M RA, resulting in an 80.1% wound healing compared to 38.8% in the control (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0002). Treatment with 5 \u0026micro;M RA led to 67.2% wound closure (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0341), while 3 \u0026micro;M RA increased closure to 61.0%, though this was not statistically significant. HUVECs cultured in complete endothelial growth media (EGM) with 3% foetal calf serum (FCS), serving as a positive control, exhibited high wound healing capacity (86.1%) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F). However, higher RA concentrations (20 \u0026micro;M and 50 \u0026micro;M) were toxic, leading to cell death after 19 hours of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eWe also confirmed that consistent with previous findings (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), direct treatment of the alveolar epithelial cell line A549 with RA did not induce wound healing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). A549 cells cultured in DMEM supplemented with 10% foetal bovine serum (FBS) served as a positive control, demonstrating nearly complete wound closure after 19 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e\n\u003ch3\u003eTranscriptomic profiling of RA-induced responses in HPMECs\u003c/h3\u003e\n\u003cp\u003eTo investigate the mechanisms underlying RA-induced angiogenesis in HPMECs, transcriptomic profiling was performed to examine gene expression changes during this process. Time-lapse imaging of angiogenesis was first conducted to determine critical time points for further analysis. As shown in Supplementary Video S1, RA-treated HPMECs rapidly migrated and reorganised into tube-like structures within 40 minutes of seeding on Matrigel, whereas vehicle-treated (EtOH) controls remained randomly distributed at the same time point (Video S2). By 60 minutes, clear differences in tube formation were evident between control and RA-treated cells (Videos S1 and S2). By 200 minutes, extensive tube networks had formed in RA-treated HPMECs. Based on these observations, RNA was extracted at 40 min, when tube initiation was first apparent, and at 4 hr, when significant networks were evident, for whole-genome expression analysis comparing EtOH control versus RA-treated HPMECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMicroarray analysis identified 962 differentially expressed genes (DEGs) at 40 minutes and 545 DEGs at 4 hours post-treatment between control and RA-treated HPMECs [fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 or \u0026lt; -1.5, false detection rate (FDR)-adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05] (Supplementary Tables S1 and S2). Notably, volcano plot analysis highlighted key components of the RA signalling pathway, including \u003cem\u003eRARB\u003c/em\u003e (retinoic acid receptor beta), \u003cem\u003eDHRS3\u003c/em\u003e (dehydrogenase/reductase 3), \u003cem\u003eNRIP1\u003c/em\u003e (nuclear receptor interacting protein 1), and \u003cem\u003eRDH10\u003c/em\u003e (retinol dehydrogenase 10), among the most upregulated genes at 4 hours post-RA treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C). In contrast, none of the top genes upregulated at 40 minutes were related to RA signalling (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRA is the primary ligand for RAR-RXR heterodimers, which bind to retinoic acid response elements (RAREs) to initiate transcription of RA target genes (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). To further investigate the response of HPMECs to RA, the top 15 DEGs at both the 40-minute and 4-hour time points were analysed to determine whether these genes contained a RARE motif (Supplementary Tables S3 and S4). \u003cem\u003eIn silico\u003c/em\u003e prediction of RARE DR5 sequences was performed using oPOSSUM3, and results were cross-referenced with previously published RARE-containing genes (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Interestingly, the top 15 upregulated genes at 4 hours showed significant enrichment for RARE-containing genes (47%; 7 out of 15 genes), including \u003cem\u003eDHRS3, RARB, NRIP1, HIC1, HOXA3, HOXA5, and ARHGAP18\u003c/em\u003e (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). In contrast, none of the top 15 upregulated genes at 40 minutes contained RARE sequences (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). These findings strongly suggest that a cellular response to exogenous RA has occurred by 4 hours post-treatment, leading us to focus our subsequent gene enrichment and functional analyses on the 4-hour dataset.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that RA induces the upregulation of vascular endothelial growth factor A (\u003cem\u003eVEGFA\u003c/em\u003e) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Consistent with this, our findings showed increased \u003cem\u003eVEGFA\u003c/em\u003e expression at 40 minutes and upregulation of vascular endothelial growth factor receptor 1 (\u003cem\u003eVEGFR1\u003c/em\u003e or \u003cem\u003eFLT1\u003c/em\u003e) at 4 hours post-RA treatment. No changes were found in other genes involved in VEGF signalling, including \u003cem\u003eVEGFB\u003c/em\u003e, \u003cem\u003eVEGFC, FLT4 (VEGFR3), KDR (VEGFR2)\u003c/em\u003e, neuropilin-1 (\u003cem\u003eNRP1\u003c/em\u003e), and neuropilin-2 (\u003cem\u003eNRP2\u003c/em\u003e) (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eEnriched GO terms and pathways in RA-treated HPMECs\u003c/h3\u003e\n\u003cp\u003eSince cellular responses to RA have occurred by 4 hours post-treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C, and Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), we performed gene set enrichment analysis (GSEA) on DEGs from this time point using Panther and Cytoscape. Several key biological processes were significantly enriched in RA-treated HPMECs. The most prominent processes included regulation of cell adhesion, developmental processes, and actin filament organisation, all highly significant after FDR adjustment. Additional enriched processes, including epithelial cell proliferation, differentiation, cell motility, response to wounding, and tube morphogenesis, pointed to a robust cellular response to RA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePathway enrichment analysis further confirmed activation of RA signalling in RA-treated HPMECs. Pathways directly related to RA, including retinoic acid signalling and RA biosynthesis, were significantly enriched at 4 hours post-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, pathways such as interferon signalling and CCKR signalling suggest that RA also modulates immune responses. Notably, the EGF receptor (EGFR) signalling pathway, which has been implicated in lung development(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and various chronic pulmonary diseases, including lung fibrosis, COPD, asthma, and lung cancer (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), as well as lung repair (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), was also significantly enriched.\u003c/p\u003e\n\u003ch3\u003eIdentifying secreted proteins upregulated in RA-treated HPMECs\u003c/h3\u003e\n\u003cp\u003eLung repair requires coordinated crosstalk among alveolar cell types (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Since RA promoted angiogenesis and wound healing in endothelial cells but had no direct effect on alveolar epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we hypothesised that RA signalling may induce the release of pro-repair signals from the endothelium to act in a paracrine manner on other alveolar cell populations. To identify candidate secreted factors, the 545 DEGs detected at 4 hours post RA-treatment were subjected to Panther \u0026ldquo;Protein classification\u0026rdquo; analysis. This revealed 14 candidate genes encoding intercellular signalling proteins, eight of which were upregulated, including \u003cem\u003eCXCL8\u003c/em\u003e (C-X-C motif chemokine ligand 8), \u003cem\u003eTGFA\u003c/em\u003e (transforming growth factor alpha\u003cb\u003e)\u003c/b\u003e, \u003cem\u003eBMP2\u003c/em\u003e (bone morphogenetic protein 2), \u003cem\u003eFGF20\u003c/em\u003e (fibroblast growth factor 20), \u003cem\u003eWNT9A\u003c/em\u003e (Wnt family member 9a), \u003cem\u003eGDF15\u003c/em\u003e (growth differentiation factor 15), \u003cem\u003eEFNB1\u003c/em\u003e (Ephrin B1), and \u003cem\u003eADM\u003c/em\u003e (adrenomedullin) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Three genes, \u003cem\u003eCXCL8, TGFA\u003c/em\u003e, and \u003cem\u003eBMP2\u003c/em\u003e, were selected for further analysis due to their known association with lung diseases and relevant biological functions (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Notably, TGFα, encoded by \u003cem\u003eTGFA\u003c/em\u003e, is a ligand for the EGFR signalling pathway, one of the top enriched pathways in RA-treated HPMECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIdentification of 14 genes encoding intercellular signalling proteins.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene symbol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProtein class\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMapped to signalling pathway\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFold Change\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFDR\u003c/p\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCXCL8\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003echemokine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCCKR signalling map, Interleukin signalling pathway, Inflammation mediated by chemokine and cytokine signalling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.56E-07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTGFA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003egrowth factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEGF receptor signalling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0176\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBMP2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003egrowth factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGF-beta signalling pathway, Gonadotropin-releasing hormone receptor pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eFGF20\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003egrowth factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFGF signalling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0184\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eWNT9A\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eintercellular signal molecule\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWnt signalling pathway, cadherin signalling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0376\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGDF15\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003egrowth factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGF-beta signalling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0087\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEFNB1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emembrane-bound signalling molecule\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAngiogenesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0286\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eADM\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epeptide hormone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0303\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCCL23\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecytokine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-9.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0188\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eWNT5A\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eintercellular signal molecule\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAngiogenesis, Wnt signalling pathway, cadherin signalling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-8.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0167\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePDYN\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eneuropeptide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEnkephalin release, Opioid prodynorphin pathway, Opioid proenkephalin pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-4.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0241\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePTN\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003egrowth factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-3.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0473\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eFGF5\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003egrowth factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFGF signalling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-2.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0129\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eFGF8\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003egrowth factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFGF signalling pathway\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-2.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0484\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eInterestingly, qRT-PCR validation confirmed the upregulation of \u003cem\u003eBMP2\u003c/em\u003e and \u003cem\u003eCXCL8\u003c/em\u003e in RA-treated HPMECs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), consistent with the microarray data, but no significant changes were observed in \u003cem\u003eTGFA\u003c/em\u003e transcript levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Additional top upregulated genes, \u003cem\u003eHOXA3\u003c/em\u003e and \u003cem\u003eHOXA5\u003c/em\u003e, were also validated by qRT-PCR, corroborating the microarray results (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). It is important to note that RNA for qRT-PCR validation was isolated from RA-treated HPMEC monolayers cultured on fibronectin-coated dishes, whereas RNA for the microarray analysis was obtained from RA-treated HPMECs undergoing angiogenesis.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eTGFα induces wound healing and cell migration in alveolar epithelial cells\u003c/h2\u003e\u003cp\u003eTo evaluate the effects of TGFα, BMP2, and CXCL8 on wound healing and cell migration in alveolar epithelial cells, we treated A549 cells and primary human alveolar type 2 (hAT2) epithelial cells with recombinant proteins and performed scratch wound and cell migration assays, respectively. Treatment with TGFα significantly enhanced wound healing in A549 cells in a dose-dependent manner, with the maximal effect observed at 40 ng/ml (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to the untreated control (DMEM supplemented with 0.5% FBS). Treatment with 4 ng/ml TGFα also significantly increased wound closure (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0149) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Additionally, immunostaining with the proliferation marker Ki67, followed by quantification of Ki67-positive cells, revealed no significant differences in cell proliferation across the various concentrations of TGFα (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). MTT assays further confirmed that TGFα did not affect cell viability at any concentrations tested. Cells treated with 70% methanol were used as a positive control for dead cells, which exhibited significant cell death compared to the untreated control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003) (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we replicated the scratch assay in human primary alveolar type 2 (hAT2) cells isolated from resected lung tissue, following a previously described method (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). hAT2 cells exhibited low wound closure (10.5% to 20.8%) (Figure S5), likely due to the removal of collagen-I matrix in the scratch area, which impaired hAT2 migratory capacity. We then employed a cell migration assay to further assess the effect of TGFα on hAT2 cells. Based on the dosage-response observed in the scratch assay (Figure S5), we narrowed the range of TGFα concentrations for subsequent experiments to 0.2 ng/ml to 4 ng/ml. Consistent with findings in A549 cells, TGFα treatment enhanced hAT2 migration in a concentration-dependent manner, with the most pronounced effect observed at 4 ng/ml TGFα (42.5% cell migration rate) compared to 26.3% in the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0314) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and E). TGFα treatment also did not significantly impact cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), and labelling of hAT2 cells with NucView 488-caspase-3 dye indicated that TGFα did not induce apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). MTT assays confirmed that cell viability was maintained in TGFα-treated cells. Cells treated with 70% methanol served as a positive control for dead cells, demonstrating a significant reduction in cell viability compared to the untreated control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Figure S6).\u003c/p\u003e\u003cp\u003eThe effects of BMP2 and CXCL8 on wound healing in A549 cells were also investigated; however, no significant differences were observed between treated and untreated cells at any concentrations tested (Figures S7A and B). MTT assays confirmed the concentrations of BMP2 and CXCL8 used in these experiments did not affect cell viability (Figures S7C and D).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTGFα does not modulate cell migration or proliferation in human lung fibroblasts and microvascular endothelial cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine whether TGFα also influences cell migration in other alveolar cell populations, cell migration and scratch wound assays were performed on primary human lung fibroblasts (HLFs) and HPMECs, respectively. Our results demonstrated that TGFα did not significantly affect cell migration in HLFs (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). Furthermore, immunostaining for the proliferation marker Ki67 revealed no effect of TGFα on cell proliferation. HLFs cultured in DMEM supplemented with 10% FBS exhibited a significantly higher percentage of Ki67-positive cells (42.9%) compared to control cells grown in DMEM containing reduced FBS (25.7%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0172) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, no apoptotic cells were detected in either control or TGFα-treated HLFs, whereas cells treated with 70% methanol served as a positive control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Similarly, TGFα had no effect on wound healing in HPMECs (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F). Cell viability assays confirmed that TGFα treatment did not influence metabolic activities in either HLFs or HPMECs (Figures S8A and B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn summary, our findings demonstrate that the pro-migratory and wound healing effects of TGFα are specific to alveolar epithelial cells.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEndothelial angiogenesis-induced TGFα modulates epithelial wound healing through activation of EGFR signalling\u003c/h3\u003e\n\u003cp\u003eGiven that TGFα is a well-established ligand for EGFR signalling, we next investigated whether TGFα-induced epithelial wound healing is mediated through this pathway. First, to establish that EGFR activation could be reliably detected, A549 cells were treated with recombinant TGFα for 30 minutes or 2 hours and immunostained with an anti-phospho-EGFR antibody. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, there was no noticeable difference in phospho-EGFR staining between the serum-free controls and TGFα-treated A549 cells after 30 minutes. Epidermal growth factor (EGF), another EGFR ligand, served as a positive control but similarly showed no change in EGFR staining at this early time point. However, after 2 hours, changes in the pattern of phospho-EGFR were observed in both TGFα- and EGF-treated cells, particularly at the cell membrane, which were not present in control cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and C). This suggests that EGFR activation is detectable at 2 hours after TGFα stimulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether TGFα secreted by endothelial cells during angiogenesis could activate EGFR in A549 cells, we employed a transwell co-culture system in which A549 cells were cultured in the upper chamber and HPMECs, treated with either RA or vehicle control (EtOH), were seeded on Matrigel in the lower chamber (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Remarkably, A549 cells co-cultured with RA-treated HPMECs showed marked changes in the localisation of phospho-EGFR staining compared to those co-cultured with EtOH-treated HPMECs (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and F). These findings indicate that RA-induced angiogenesis in HPMECs activates EGF receptor in adjacent epithelial cells.\u003c/p\u003e\u003cp\u003eTo assess whether extracellular TGFα secreted from HPMECs contributed to this effect, we measured TGFα levels in conditioned media collected from EtOH- or RA-treated HPMECs at 4 and 16 hours post-treatment using ELISA. However, TGFα was undetectable in all samples. It should be noted that although the TGFα standard provided with the ELISA kit produced a reliable standard curve, media supplemented with the commercially available recombinant TGFα used in this study also yielded no detectable signal (Table S5). We were therefore unable to confirm the presence of secreted TGFα following RA treatment in this study.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we conducted a microarray to identify secreted proteins that were upregulated in RA-treated HPMECs undergoing angiogenesis. The top three most upregulated of these proteins, CXCL8, TGFa and BMP2, were selected for further investigation. TGFa specifically induced repair and migration of both A549 and human primary AT2 cells, whereas CXCL2 and BMP2 did not. In contrast, TGFa had no effect on repair or proliferation of either primary HLFs or HPMECs. Subsequently, using a co-culture approach, we found that RA treatment induced HPMECs to undergo angiogenesis, which led to activation of the TGFa receptor EGFR, in adjacent A549 cells, indicative of a paracrine effect of RA.\u003c/p\u003e\u003cp\u003eIt has long been appreciated that RA is a powerful regenerative agent across organs and species (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Interestingly, a recent study in the skin identified a pivotal role of RA in regulating stem cell fate during wound repair by modulating lineage plasticity; a transient state that bestows tissue stem cells with fate flexibility and is important in the repair response to injury (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In the lungs, RA is critical for development, but it also has a powerful ability to regenerate lung alveoli (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Epidemiological studies further demonstrate that Vitamin A deficiency has detrimental effects not only on the lungs but also other organ systems (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Promising data from animal experiments and population studies led to clinical trials being conducted with the aim of determining whether RA might be an effective treatment for patients with emphysema. Although the results of these first human lung regenerative clinical trials were largely disappointing and failed to meet their primary end points, in one trial, involving patients with CT-confirmed smoking-related emphysema, post-hoc subgroup analysis found that patients with predominantly lower-zone disease experienced reduced declines in FEV1, gas diffusion, and exercise capacity compared with placebo (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). These findings highlight the need for deeper mechanistic understanding of the cellular and molecular mechanisms through which putative pro-repair factors such as RA exert their effects prior to clinical trials taking place.\u003c/p\u003e\u003cp\u003eWe previously showed that instead of directly inducing epithelial lung repair, RA induced angiogenesis of HPMECs (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and we therefore hypothesised that RA might indirectly mediate alveolar epithelial repair in a paracrine manner by inducing secretion of a pro-repair signalling molecule(s) during angiogenesis. To investigate this, we combined the use of human and 3D \u003cem\u003eex vivo\u003c/em\u003e models with detailed mechanistic studies of RA driven lung repair. Our findings show that in support of our hypothesis, following RA-induced angiogenesis of HPMECs, TGFα is upregulated and this secreted growth factor specifically drives repair of human alveolar epithelial cells.\u003c/p\u003e\u003cp\u003eTGFα is one of seven ligands that bind to EGFR with varying affinities (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Considerable evidence has demonstrated a role for EGF ligands and EGFR signalling in lung repair (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). However, although TGFα is expressed in the lungs throughout the life course, its role in repair and regeneration remains unclear. Instead, adult lung studies have focused on the detrimental effects of aberrant TGFα expression. For example, overexpression of TGFα in mice leads to enlarged alveolar airspaces and fibrosis, although lower doses did not produce significant changes (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Interestingly, TGFα is also upregulated in lung diseases characterised by tissue damage, including idiopathic pulmonary fibrosis (IPF), COPD, and in post-COVID patients (\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). While this upregulation has often been interpreted as detrimental, it is equally plausible that increased expression reflects an adaptive repair response, with TGFα contributing positively to lung regeneration. Recently, Li and colleagues showed that lipopolysaccharide (LPS) injury induces upregulation of TGFα/EGFR signalling, leading to abnormal elastin deposition and alveolar simplification; inhibition of EGFR with erlotinib mitigated these effects on alveoli, underscoring the need for tight regulation of TGFα levels and highlighting the TGFα-EGFR axis as a potential therapeutic target to modulate alveolar regeneration (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFrom our microarray data, we identified eight upregulated genes encoding secreted proteins, of which the top three were selected for detailed investigation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Strikingly, only TGFα promoted epithelial repair. Furthermore, the pro-repair capacity of TGFα is specific to lung alveolar epithelial cells, as no effect was observed on the repair of human lung fibroblasts or HPMECs.\u003c/p\u003e\u003cp\u003eIntriguingly, unlike the other candidate signalling molecules, TGFα mRNA was not upregulated in monolayers of RA-treated HPMECs but was increased in RA-treated HPMECs undergoing angiogenesis. This suggests that \u003cem\u003eTGFA\u003c/em\u003e induction is not a direct consequence of RA treatment but rather a response stimulated during RA-induced angiogenesis, thereby supporting our hypothesis that a paracrine signal released following HPMEC angiogenesis drives epithelial repair. This contrasts with a previous study reporting suppression of \u003cem\u003eTGFA\u003c/em\u003e expression in upper airway epithelial cells and airway explants treated with retinol (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Notably, our study used retinoic acid (a derivative of retinol) at 10 \u0026micro;M compared with 0.1 \u0026micro;M retinol in Miller\u0026rsquo;s work and focused on alveolar cells rather than airway epithelial cells or tissues, suggesting that \u003cem\u003eTGFA\u003c/em\u003e transcriptional responses to retinoids are tightly regulated, both dose- and context-dependently.\u003c/p\u003e\u003cp\u003eThe validity of the microarray data was confirmed by the demonstration that RA signalling was among the significantly enriched pathways in RA-treated HPMECs at 4 hr. GO enrichment analysis highlighted two main aspects of RA-induced changes: first, reorganisation of actin filaments, which governs cell adhesion and migration\u0026mdash;processes essential for wound healing and tissue repair; and second, changes in epithelial cell behaviour, supporting the idea of crosstalk between HPMECs and epithelial cells in response to exogenous RA exposure.\u003c/p\u003e\u003cp\u003eAlthough we identified a pro-repair effect of TGFα on epithelial cells and demonstrated EGFR activation in RA-treated A549 cells co-cultured with HPMECs undergoing angiogenesis, we were unable to detect TGFα in supernatants collected from these co-cultures at either 4 or 16 hours. The ELISA kit was validated with a standard curve, but no TGFα was detected in the co-culture supernatants, or even in culture media supplemented with recombinant TGFα, which was intended to serve as a positive control (Table S5). This may reflect methodological limitations, such as suboptimal timing of supernatant collection or insufficient recognition of specific TGFα isoform(s) by the antibodies used. Notably, Ensembl reports multiple isoforms of TGFα, which could contribute to antigen-antibody mismatch.\u003c/p\u003e\u003cp\u003eDespite this limitation, we were able to show activation of EGFR signalling in A549 cells in response to RA-treated HPMECs undergoing angiogenesis in co-culture. Ideally, we would have further confirmed this by blocking a TGFα-specific EGFR phosphorylation site. However, no known TGFα-specific EGFR phosphorylation sites have been identified to date. Direct inhibition of TGFα could be considered, but this strategy risks confounding results, as the absence of TGFα may trigger compensatory activity from other EGFR ligands. Indeed, experimental evidence showed that loss of individual EGFR ligands, including TGFα, can be functionally compensated for by others, and only simultaneous inhibition of multiple ligands substantially reduces EGFR signalling (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn summary, this study shows that RA induces angiogenesis in lung endothelial cells, leading to TGFα-dependent activation of EGFR, which drives repair of alveolar epithelial cells. More broadly, our findings highlight the importance of using physiologically relevant human models to uncover the detailed mechanisms of tissue repair required for the development of effective pharmacological regenerative medicine treatments.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003eSex as a biological variable\u003c/h2\u003e\n \u003cp\u003eHuman tissue, for cell isolation, was obtained from both males and females.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy approval\u003c/h2\u003e\n \u003cp\u003eLung tissue samples were collected from male and female patients undergoing lung resection at the Royal Brompton \u0026amp; Harefield hospital (RBHT), by Biobank sample co-ordinators within the RBHT Clinical Research Facility (CRF). All samples were obtained following informed consent from patients. The research was conducted using the RBHT Biobank ethics number: 20/SC/0142.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eMice\u003c/h2\u003e\n \u003cp\u003eAll animal maintenance and procedures were conducted in compliance with the requirements of the Animal (Scientific Procedures) Act 1986. Animal work was approved by the South Kensington AWERB committee at Imperial College London. Mice were housed in specific pathogen-free conditions and given food and water \u003cem\u003ead libitum\u003c/em\u003e. Wild-type female mice aged 10\u0026ndash;12 weeks were purchased from Charles River (United Kingdom).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eMouse Precision-Cut Lung Slices (PCLS) and acid injury\u003c/h2\u003e\n \u003cp\u003eMouse PCLS were generated following previously established protocols (\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e). Briefly, adult mice were euthanised via intraperitoneal injection of pentobarbital. The anterior chest and neck wall were carefully dissected, and a small incision was made in the trachea below the cricoid cartilage. A 21G rigid metal cannula was inserted into the trachea incision, and the lungs were inflated by injecting 1.2 ml of 2% (w/v) low-gelling agarose (Sigma\u0026ndash;Aldrich, A9414) dissolved in 1\u0026times; Hank\u0026rsquo;s Balanced Salt Solution (HBSS; Life Technology, 14025100) containing 1% HEPES buffer (Life Technology, 15630080). The chest cavity was cooled with ice to solidify the agarose.\u003c/p\u003e\n \u003cp\u003eThe lungs and heart were excised and submerged in ice-cold HBSS/HEPES buffer. Lung lobes were carefully separated and sliced transversely at a thickness of 300 \u0026micro;m using an automated vibratome (Compresstome VF-300-0Z; Precisionary Instruments LLC) in ice-cold HBSS/HEPES buffer. PCLS were then transferred to 24-well plates containing ice-cold serum-free (SF) Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM)-GlutaMAX (Life Technologies, 31966021) with 1% penicillin-streptomycin (Merck, P0781). PCLS were incubated overnight at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere and washed three times with warm SF-DMEM to remove any remaining agarose.\u003c/p\u003e\n \u003cp\u003eFor acid injury, PCLS were treated with 0.1 M HCl (Fisher Chemical, 10053023) for 1 minute at room temperature (RT). The HCl solution was then discarded, and PCLS were washed three times with phosphate-buffered saline (PBS; Sigma; D8537) to remove residual acid. PCLS were cultured in fresh SF-DMEM for 48 hours before RNA extraction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eCell culture\u003c/h2\u003e\n \u003cp\u003ePrimary human pulmonary microvascular endothelial cells (HPMECs; Promocell, C12281) and human umbilical vein endothelial cells (HUVECs; Promocell, C12200) were cultured in Endothelial Cell Growth Medium MV (EGM-MV; Promocell, C22020) and Endothelial Growth Media (EGM; Promocell, C-22010), respectively. Cells were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Cells were used between passages 3 and 7, showing no changes in morphology or baseline tube-forming capacity. HPMECs used in this study were sourced from two Caucasian female donors, aged 64 and 98. A549 cells, a human alveolar adenocarcinoma epithelial cell line, were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM-GlutaMAX (Gibco) supplemented with 10% foetal bovine serum (FBS; Sigma, F9665) and 1% penicillin/streptomycin at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003ePrimary human alveolar type 2 (hAT2) epithelial cells and human lung fibroblasts (HLFs) were isolated from histologically normal lung tissues obtained from patients undergoing surgical resection for suspected lung tumours, through the Clinical Research Facility (CRF) Respiratory Biobank at Royal Brompton \u0026amp; Harefield Hospitals (Guy\u0026rsquo;s and St Thomas\u0026rsquo; NHS Foundation Trust) (Ethics reference number: 20/SC/0142). The isolation protocol was based on a previously described method (\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e) with slight modifications.\u003c/p\u003e\n \u003cp\u003eBriefly, lung tissue was perfused with HBSS (Invitrogen, 14025-050) to remove blood and debris, followed by digestion with 0.25% trypsin (Sigma, T8003) at 37\u0026deg;C for 45 minutes. Trypsinisation was halted by adding FBS, and the tissue was finely chopped. DNase I (Sigma, DN25) was added to facilitate degradation of extracellular DNA to improve tissue dissociation. The resulting tissue suspension was passed through a tea strainer and a 40-\u0026micro;m mesh filter (Marathon Lab Supplies, 352340) to remove undigested tissue. The filtrate was centrifuged at 1,300 rpm for 10 minutes at 12\u0026deg;C. The pellet was resuspended in hybridoma serum-free medium (SFM; Gibco, 11570416) containing 50 \u0026micro;g/ml DNase I and incubated at 37\u0026deg;C for 45 minutes to remove immune cells.\u003c/p\u003e\n \u003cp\u003eAfter incubation, the supernatant was collected and centrifuged again at 1,300 rpm for 10 minutes at 12\u0026deg;C. The pellet was resuspended in hybridoma medium containing 10% FBS and incubated at 37\u0026deg;C for 2 hours. Cells adhering to the dishes after this period were identified as HLFs, which were subsequently cultured in DMEM-GlutaMAX supplemented with 10% FBS for downstream experiments. The supernatant containing non-adherent cells was centrifuged at 1,300 rpm for 10 minutes at 12\u0026deg;C, and the resulting pellet, containing hAT2 cells, was resuspended and cultured in hybridoma medium with 10% FBS. hAT2 cells were not passaged, and all experiments involving these cells were completed within 68 hours of seeding. HLFs were used between passages 3\u0026ndash;7 for experiments.\u003c/p\u003e\n \u003cp\u003eThe specificity of isolated hAT2 cell populations was confirmed by immunostaining with alveolar cell type-specific markers. The hAT2 cells were positive for pan-cytokeratin (a pan-epithelial marker) and Pro-SPC (an AT2 cell marker), but negative for vimentin (a fibroblast marker) and podoplanin (PDPN; an AT1 cell marker) (Figure S9).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003ePharmacological treatment\u003c/h2\u003e\n \u003cp\u003eRetinoic acid (RA; Sigma, R2625) used in this study was dissolved in absolute ethanol (EtOH; Sigma-Aldrich, 51976). Recombinant human TGF\u0026alpha; (catalogue number: 239-A1-100), BMP2 (catalogue number: 355-BM-010), and CXCL8 (catalogue number: 208-IL-010) were obtained from R\u0026amp;D Systems and reconstituted according to the manufacturer\u0026rsquo;s instructions. Human EGF was sourced from Promocell (catalogue number: C-39220).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eMatrigel-based angiogenesis assay and tube quantification\u003c/h2\u003e\n \u003cp\u003eAngiogenesis \u0026micro;-slides (Ibidi, 81506) or 24-well plates (Ibidi, 2401) were pre-coated with growth factor-reduced Matrigel (BD Biosciences, 354230), diluted to a concentration of 5 mg/ml with pre-chilled endothelial cell basal medium (EBM; Promocell, C-22220) without supplements. The plates were incubated at 37\u0026deg;C for 1 hour to allow the Matrigel to solidify. HPMECs were serum-starved for 24 hours prior to conducting the tube formation assays. Cells were seeded at a density of 4,750 cells per well for the angiogenesis \u0026micro;-slides and 70,000 cells per well for the 24-well plates. Cells were treated with either the vehicle (EtOH) or 10 \u0026micro;M RA.\u003c/p\u003e\n \u003cp\u003eFor time-lapse imaging of tube formation, the \u0026micro;-slide was immediately transferred to a pre-equilibrated, humidified incubator chamber (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, and approximately 21% oxygen levels) of an inverted Zeiss Axio Observer widefield epifluorescence microscope. Imaging was performed over a 4-hour period using an EC Plan-Neofluar 10\u0026times;/0.30 objective lens. The total tube length and the total number of nodes per field of view were quantified using FIJI software. Data were expressed as the mean total tube length or total number of nodes per field of view.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e\n \u003cp\u003eMouse PCLS were homogenised using the FastPrep-24TM Tissue Homogeniser (MP Biomedicals), and total RNA was extracted with the RNeasy mini kit (Qiagen, 74104) following the manufacturer\u0026rsquo;s protocols. For each experimental condition, RNA samples were pooled from three PCLS, and a total of three RNA samples were prepared per condition. Additionally, RNA was extracted from HPMEC monolayers using the same RNeasy mini kit. RNA concentration and quality were assessed using the TapeStation 2200 (Agilent), and only samples with an RNA integrity number (RIN) greater than 8 were used for downstream cDNA synthesis and qRT-PCR.\u003c/p\u003e\n \u003cp\u003eApproximately 200 ng of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to manufacturer\u0026rsquo;s instructions. Quantitative RT-PCR was then performed using TaqMan Fast Advanced Master Mix (Life Technologies) on a 7500 Fast Real-Time PCR system (Applied Biosystems). The housekeeping gene \u003cem\u003eB2m\u003c/em\u003e was used as a reference for normalisation. Relative gene expression levels were calculated using the 2\u0026thinsp;\u0026minus;\u0026thinsp;\u0026Delta;\u0026Delta;CT method. Each sample was tested in triplicate, with four biological replicates derived from four different mice.\u003c/p\u003e\n \u003cp\u003eThe following TaqMan primers (Life Technologies) were used in this study: mouse \u003cem\u003eB2m\u003c/em\u003e (Mm00437762_m1), human \u003cem\u003eB2M\u003c/em\u003e (Hs00984230_m1), \u003cem\u003eRara\u003c/em\u003e (Mm01296312_m1), \u003cem\u003eRarb\u003c/em\u003e (Mm01319677_m1), \u003cem\u003eRarg\u003c/em\u003e (Mm00441083_m1), \u003cem\u003eAldh1a1\u003c/em\u003e (Mm00657317_m1), \u003cem\u003eAldh1a2\u003c/em\u003e (Mm00501306_m1), \u003cem\u003eAldh1a3\u003c/em\u003e (Mm00474049_m1), \u003cem\u003eCyp26a1\u003c/em\u003e (Mm00514486_m1), \u003cem\u003eCyp26b1\u003c/em\u003e (Mm00558507_m1), \u003cem\u003eCyp26c1\u003c/em\u003e (Mm03412454_m1), \u003cem\u003eTGFA\u003c/em\u003e (Hs00608187_m1), \u003cem\u003eCXCL8\u003c/em\u003e (Hs00174103_m1), \u003cem\u003eBMP2\u003c/em\u003e (Hs00154192_m1), \u003cem\u003eHOXA3\u003c/em\u003e (Hs00601076_m1), and \u003cem\u003eHOXA5\u003c/em\u003e (Hs00430330_m1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eMicroarrays and raw data processing\u003c/h2\u003e\n \u003cp\u003eMicroarray service was provided by UK Bioinformatics Ltd. (UKB, UK). Total RNA was extracted from HPMECs cultured on Matrigel using Trizol reagent (Invitrogen, UK). RNA quality was assessed using the Agilent 2100 Bioanalyzer. For each treatment and time point, four RNA samples were prepared from independent experiments, with each sample pooled from three technical replicates (see Table S6 for sample details).\u003c/p\u003e\n \u003cp\u003ecDNA was synthesised using the Ovation\u0026reg; Pico WTA System V2 and labelled with the Encore Biotin Module, following the manufacturer\u0026rsquo;s protocols (Nugen, USA). The labelled cDNA was hybridised to Affymetrix Human U133 Plus 2.0 Gene Chips (Affymetrix) according to the manufacturer\u0026rsquo;s instructions. The resulting microarray data were generated in an Affymetrix CEL format and processed using the Transcriptome Analysis Console (TAC) software v4.0.2 (Thermo Scientific).\u003c/p\u003e\n \u003cp\u003eRaw data normalisation and summarisation were performed using the MAS5 algorithm within the TAC software. Gene expression analysis was conducted using the human genome reference assembly hg19. Differential expression was defined by a fold change threshold of \u0026gt;\u0026thinsp;1.5 or \u0026lt; -1.5, and a false discovery rate (FDR)-adjusted \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05. Statistical significance was calculated using moderated t-statistics from the empirical Bayes (ebayes) method within an ANOVA framework, allowing for robust identification of differentially expressed genes between RA-treated and EtOH-treated control samples. FDR correction was applied using the Benjamini-Hochberg algorithm to control for multiple testing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eGene Set Enrichment Analysis (GSEA)\u003c/h2\u003e\n \u003cp\u003eGSEA was performed to identify pathways and biological processes significantly associated with upregulated genes. Pathway enrichment analysis was conducted using PANTHER version 19.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.pantherdb.org/\u003c/span\u003e\u003c/span\u003e), focusing on PANTHER Pathways and Reactome Pathways. For the enrichment of Gene Ontology (GO) biological processes, Cytoscape software version 3.10.2 was utilised, in conjunction with the STRING enrichment tool. Enriched pathways and biological processes were ranked according to their statistical significance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eScratch wound and cell migration assays\u003c/h2\u003e\n \u003cp\u003eFor the scratch wound assay, 45,000 HUVECs, HPMECs, or A549 cells were seeded onto 8-well Permanox chamber slides (Fisher Scientific; 16250681), pre-coated with 2 \u0026micro;g/ml fibronectin (Sigma, F1141) for HUVECs and HPMECs, or 50 \u0026micro;g/ml rat-tail collagen I (Gibco, A1048301) for A549 cells. Cells were cultured in their respective complete media - EGM for HUVECs, EGM-MV for HPMECs, and DMEM-GlutaMAX supplemented with 10% FBS for A549, and incubated overnight at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e to reach confluence. A scratch wound was made across the centre of the confluent monolayer using a P1000 pipette tip. Wells were gently rinsed with PBS to remove dislodged cells, followed by the addition of appropriate treatments.\u003c/p\u003e\n \u003cp\u003eFor RA treatments, HUVECs were exposed to 3 \u0026micro;M, 5 \u0026micro;M, 10 \u0026micro;M, 20 \u0026micro;M, or 50 \u0026micro;M RA, diluted in EBM. A549 cells were treated with 10 \u0026micro;M of RA in SF-DMEM. Since RA was reconstituted in EtOH, control media containing 0.1% EtOH in either EBM or SF-DMEM were used for HUVECs and A549 cells, respectively. Positive control media for enhanced wound healing consisted of EGM with 3% FCS for HUVECs and DMEM supplemented with 10% FBS for A549 cells.\u003c/p\u003e\n \u003cp\u003eFor the cell migration assay, hAT2 and HLFs were plated using 2-well silicone culture inserts (Ibidi, 80209) adhered to 50 \u0026micro;g/ml rat-tail collagen I-coated 13 mm coverslips placed in 24-well plates. Freshly isolated hAT2 cells were seeded at a density of 250,000 cells per well, while HLFs were plated at 15,000 cells per well. For HLFs, the silicone inserts were removed after overnight incubation. For hAT2 cells, debris and dead cells were gently washed off with PBS 24 hours post-seeding, and fresh hybridoma SFM supplemented with 10% FBS was added, followed by a further 24-hour incubation before insert removal. TGF\u0026alpha; treatments were administered at appropriate concentrations in DMEM with 1% FBS for HLFs or in hybridoma SFM with 1% FBS for hAT2 cells.\u003c/p\u003e\n \u003cp\u003eCells in both assays were imaged at time points \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0 h and the endpoint at \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;19 h or 24 h using an inverted Zeiss Axio Observer widefield epifluorescence microscope housed in a pre-equilibrated, humidified incubator chamber (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, ~\u0026thinsp;21% oxygen). Images were captured using an EC Plan-Neofluar 10\u0026times;/0.30 objective lens, and tiling was employed to image the cell-covered areas. Wound healing percentage was quantified using a FIJI macro developed by Steve Rothery (Facility for Imaging by Light Microscopy, Imperial College London) or through manual analysis. For manual quantification, the wound areas were outlined with the freehand tool in FIJI, and the percentage healed was calculated by subtracting the endpoint area from the area at \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0 h. Three independent experiments were conducted, and each experiment was run in triplicate.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eImmunofluorescence and imaging\u003c/h2\u003e\n \u003cp\u003eCells were fixed in 4% paraformaldehyde (PFA) for 10 minutes at RT, followed by PBS. Permeabilisation was carried out using 0.2% (v/v) Triton X-100 in PBS RT for 5 minutes at RT. Blocking was then performed for 1 hour at RT using PBSBT buffer, which contains 3% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS. After blocking, cells were incubated overnight at 4\u0026deg;C with primary antibodies diluted in PBSBT blocking buffer. The following primary antibodies were used in this study: rabbit anti-vimentin (Bioorbyt, orb304659; 1:200), mouse anti-pan-cytokeratin (Sigma\u0026ndash;Aldrich, C2931; 1:200), rabbit anti-Pro-SP-C (Abcam, ab90716; 1:300), hamster anti-PDPN (Developmental Studies Hybridoma Bank, 8.1.1; 1:50), rat anti-Ki67 (eBioscience, 14-5698-82; 1:500), and rabbit anti-phospho-EGFR Y1068 (Cell Signalling Technology, 3777S; 1:100).\u003c/p\u003e\n \u003cp\u003eFollowing three washes with PBS, cells were incubated for 1 hour at RT with rhodamine phalloidin (Biotium, 00027; 1:300) and species-specific Alexa Fluor 488 and 647 secondary antibodies (Thermo Fisher Scientific; 1:500). After further PBS washes, cell nuclei were stained with DAPI (Thermo Scientific, 62249). Coverslips were mounted with ProLong Gold Antifade Mountant (Invitrogen, P36930). Imaging was performed using a Leica SP8 inverted confocal microscope equipped with a HC PL APO 10\u0026times;/0.40 air objective lens or a HC PL APO 40\u0026times;/1.30 oil objective lens. For some experiments, channel colours were changed during image post-processing to improve visual clarity.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eMTT assay\u003c/h2\u003e\n \u003cp\u003eTo evaluate the impact of pharmacological treatments on cell viability, an MTT assay was conducted according to the manufacturer\u0026rsquo;s instructions (Roche, 11465007001). Briefly, 150 \u0026micro;l of 10% MTT solution diluted in serum-free media was added to each well of a 96-well plate and incubated at 37\u0026deg;C for 45 minutes. The resulting formazan crystals, which formed in viable cells, were solubilised by adding an equal volume of DMSO and incubating at 37\u0026deg;C for 10 minutes. 100 \u0026micro;l of eluted formazan solution was measured spectrophotometrically, with absorbance recorded at 570 nm and corrected at 690 nm using a plate reader.\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eCaspase-3 assay\u003c/h2\u003e\n \u003cp\u003eTo identify apoptotic cells, the NucView\u0026reg; 488 Caspase-3 assay kit (Biotium; 30029-T) was employed, following the manufacturer\u0026rsquo;s protocols. At the end point of pharmacological treatments, cells were incubated with 5 \u0026micro;M NucView 488 substrate (prepared as 1:40 stock dilution) in the dark at RT for 30 minutes. After incubation, the dye was removed, and cells were gently washed three times with PBS. Cells were then fixed with 4% PFA for 10 minutes at RT. After fixation, the PFA was discarded, and the cells were washed three times with PBS. Cells were counterstained with DAPI at RT for 5 minutes and washed once with PBS. Cells were then mounted using ProLong\u0026reg; Gold Antifade Reagent and imaged on the same day using a Leica SP8 inverted confocal microscope equipped with a HC PL APO 40\u0026times;/1.30 oil objective lens.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eTranswell insert-based co-culturing of HPMECs and A549\u003c/h2\u003e\n \u003cp\u003eHPMECs and A549 cells were co-cultured as illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD. A549 cells were plated at a density of 37,000 per well in DMEM supplemented with 10% FBS onto a 24-well transwell insert with 0.4-\u0026micro;m pore polyethylene terephthalate (PET) membrane (Brand, BR782711) pre-coated with 50 \u0026micro;g/ml rat tail collagen-I. The cells were incubated overnight at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The following day, HPMECs were seeded on Matrigel and maintained in EBM as detailed in the methods section on \u0026ldquo;Matrigel-based angiogenesis assay and tube quantification\u0026rdquo;. HPMECs in the lower chamber were treated with 0.1% EtOH or 10 \u0026micro;M RA.\u003c/p\u003e\n \u003cp\u003eNext, the DMEM from the transwell inserts containing A549 cells was discarded, and the cells were washed three times with PBS to remove any residual FBS. The media in the inserts was then replaced with EBM supplemented with 0.2% FCS. The transwell inserts containing A549 cells were subsequently placed onto the HPMECs, allowing for co-culture durations of 2 hours, 4 hours, or 16 hours. After 2 hours of co-culture, A549 cells in the upper chamber were fixed and immunostained with phospho-EGFR, while culture supernatants from the HPMEC were collected at the 4-hour and 16-hour time points to assess extracellular TGF\u0026alpha; levels using the Human TGF-alpha DuoSet ELISA (R\u0026amp;D Systems, DY239) following the manufacturers\u0026rsquo; instructions.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll graphs and statistical tests were produced using GraphPad Prism 10. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). The D\u0026rsquo;Agostino\u0026ndash;Pearson test was employed to assess the normality of the data. For non-parametric datasets, the Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e-test was utilised for comparisons between two groups, while comparisons involving more than two groups were performed using the Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s multiple comparison test. The specific statistical tests applied, sample size (\u003cem\u003en\u003c/em\u003e), and the number of experiments conducted are detailed in the figure legends. A \u003cem\u003ep-\u003c/em\u003evalue of less than 0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests:\u003c/h2\u003e\u003cp\u003eAll authors declare no financial or non-financial competing interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was funded by the Royal Brompton and Harefield Hospitals Charity (Grant 123/P90719) and an award from Mr and Mrs Youssef Mansour. Imaging for this study was conducted in the Facility for Imaging by Light Microscopy (FILM) at Imperial College and was part supported by Wellcome Trust (grant 104931/Z/14/Z). Funders played no role in study design, data collection, analysis, data interpretation of data, or the writing of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCD, MH and S-SC conceived and designed the study. S-SC, CY, RM and DC performed experiments and analysed data. DC and MG provided conceptual advice. S-SC and CD wrote the manuscript. All authors contributed to editing the manuscript. MH and CD acquired funding.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe microarray data generated or analysed during the current study are available in the Gene Expression Omnibus (GEO) repository under the accession number GSE307491.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBasil MC, Alysandratos KD, Kotton DN, Morrisey EE. Lung repair and regeneration: Advanced models and insights into human disease. Cell Stem Cell. 2024;31(4):439\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Y, Wang L, Ma S, Cheng L, Yu G. Repair and regeneration of the alveolar epithelium in lung injury. FASEB J. 2024;38(8):e23612.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen Y, Li Z, Ji G, Wang S, Mo C, Ding BS. Lung regeneration: diverse cell types and the therapeutic potential. 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Nutrients. 2018;10(9).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJones PW, Rames, A.D. TESRA: Treatment of Emphysema with a Gamma-selective Retinoid agonist. Am J Respir Crit Care Med. 2011;183(on line abstracts issue).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh AB, Harris RC. Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell Signal. 2005;17(10):1183\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrinh NT, Prive A, Kheir L, Bourret JC, Hijazi T, Amraei MG, et al. Involvement of KATP and KvLQT1 K\u0026thinsp;+\u0026thinsp;channels in EGF-stimulated alveolar epithelial cell repair processes. Am J Physiol Lung Cell Mol Physiol. 2007;293(4):L870-82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi J, Cao J, Yan C, Gong X. TGF-alpha/EGFR signaling promotes lipopolysaccharide-induced abnormal elastin deposition and alveolar simplification. Exp Cell Res. 2024;437(1):113997.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiller LA, Zhao YH, Wu R. Inhibition of TGF-alpha gene expression by vitamin A in airway epithelium. J Clin Invest. 1996;97(6):1429\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin S, Hirayama D, Maryu G, Matsuda K, Hino N, Deguchi E, et al. Redundant roles of EGFR ligands in the ERK activation waves during collective cell migration. Life Sci Alliance. 2022;5(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkram KM, Yates LL, Mongey R, Rothery S, Gaboriau DCA, Sanderson J, et al. Time-lapse Imaging of Alveologenesis in Mouse Precision-cut Lung Slices. Bio Protoc. 2019;9(20):e3403.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheong SS, Akram KM, Matellan C, Kim SY, Gaboriau DCA, Hind M, et al. The Planar Polarity Component VANGL2 Is a Key Regulator of Mechanosignaling. Front Cell Dev Biol. 2020;8:577201.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-regenerative-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjregenmed","sideBox":"Learn more about [npj Regenerative Medicine](http://www.nature.com/npjregenmed/)","snPcode":"41536","submissionUrl":"https://mts-npjregenmed.nature.com/cgi-bin/main.plex","title":"npj Regenerative Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TGF-alpha, retinoic acid, EGFR signalling, angiogenesis, alveolar repair, pulmonary microvascular endothelial cells, lung repair, cell migration","lastPublishedDoi":"10.21203/rs.3.rs-7704784/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7704784/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLung repair involves coordination of multicellular processes, including endothelial angiogenesis and epithelial repopulation. Retinoic acid (RA) signalling is crucial for lung development, homeostasis, and repair, however, the mechanisms through which RA drives repair are still unknown. It has previously been shown that RA has no direct effects on repair of alveolar epithelium, yet in animal studies, RA induces alveolar regeneration. Here we show that RA-induces endothelial angiogenesis which elicits paracrine effects on alveolar epithelial cells to drive repair. Transcriptomic profiling of RA-treated HPMECs undergoing angiogenesis revealed enrichment of wound healing pathways and subsequent in-silico analysis identified several secreted factors as potential mediators of paracrine pro-repair effects on the epithelium. Scratch assays demonstrated that of these secreted factors, only TGFα promoted wound healing in alveolar epithelial A549 and primary human alveolar type 2 (hAT2) cells. Further investigation determined that TGFα promoted epithelial repair by enhancing cell migration through EGFR activation, without affecting proliferation or apoptosis. Our findings identify the TGFα/EGFR axis as a key mediator of RA-induced alveolar repair and provide a potential novel therapeutic avenue to enhance alveolar regeneration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"TGF-alpha/EGFR signalling mediates retinoic acid-induced lung repair","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 11:05:32","doi":"10.21203/rs.3.rs-7704784/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-06T02:38:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T12:07:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122487508785865678277464620916395954520","date":"2025-11-13T10:55:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-12T16:54:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T16:56:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-04T11:29:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Regenerative Medicine","date":"2025-09-24T14:28:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-regenerative-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjregenmed","sideBox":"Learn more about [npj Regenerative Medicine](http://www.nature.com/npjregenmed/)","snPcode":"41536","submissionUrl":"https://mts-npjregenmed.nature.com/cgi-bin/main.plex","title":"npj Regenerative Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf0c4591-e02a-4a16-a880-f7e13041a726","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58441495,"name":"Biological sciences/Cancer"},{"id":58441496,"name":"Biological sciences/Cell biology"},{"id":58441497,"name":"Health sciences/Diseases"},{"id":58441498,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-05-19T03:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 11:05:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7704784","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7704784","identity":"rs-7704784","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}