Fibroblast growth factor-7 promotes repair of primary human bronchial epithelial cells

Fibroblast growth factor-7 promotes repair of primary human bronchial epithelial cells | 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 Research Article Fibroblast growth factor-7 promotes repair of primary human bronchial epithelial cells Skye Quinn, Alexander Jenkins, Rafaela Konstantinidi, Philip L Molyneaux, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6939652/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Fibroblast growth factor 7 (FGF7) is a potent and specific epithelial mitogen that can modulate alveolar repair however, the impact on human bronchial epithelial cells (hBECs) is limited. This study characterised repair responses of hBECs from healthy and fibrotic lungs to exogenous FGF7 supplementation. When healthy hBECs were cultured under a physiological stressor of low seeding density, treatment with recombinant human FGF7 (rhFGF7) reduced cytotoxicity, and increased survival and proliferation. Cell migration was assessed using a scratch assay, where pre-treatment of hBECs with rhFGF7 significantly increased wound closure (50.5%, p<0.001) compared to control (15.3%), accompanied by upregulation of FGF signalling genes including mTOR, PIK3CA and MAPK3. To explore modified mRNA as an alternative protein supplementation strategy for wound repair, it was found that hBECs were able to secrete dose responsive levels of FGF7 following mRNA transfection (modFGF7), and when applied to a monolayer of hBECs, wound closure was significantly improved compared to control (36.1%, p<0.05). In contrast, when hBECs from idiopathic pulmonary fibrosis (IPF) donors were cultured at low density or injured by scratch, untreated cells were capable of notable survival and wound closure (27.1%) that was not improved by rhFGF7 or modFGF7 treatment. It was found that baseline expression of genes associated with proliferation and survival including PIK3CA, AKT1 and MTOR was higher in IPF hBEC. This study demonstrates a role of FGF7 in proliferation, survival and migration of healthy hBECs but requires careful assessment dependant on disease context. Fibroblast growth factor-7 lung repair modified mRNA Human bronchial epithelial cells idiopathic pulmonary fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Messages FGF7 is an epithelial mitogen previously shown to improve repair and regeneration of oral mucosa, keratinocytes and alveolar cells. This study explores impact on human bronchial epithelial cells (hBEC). FGF7 increases proliferation, survival and migration of primary hBEC. mRNA encoding FGF7 provides an alternative approach to growth factor supplementation. Post-FGF7 treatment, genes associated with FGF7 receptor activation and epithelial repair are upregulated in healthy hBECs but not hBECs from fibrotic lung due to higher baseline levels of these genes in untreated IPF-hBECs. INTRODUCTION Fibroblast growth factor 7 (FGF7 or Keratinocyte growth factor; KGF) is a potent mitogen secreted by mesenchymal cells such as fibroblasts, and specifically targets epithelial cells by binding to its single known receptor, the IIIb isoform of FGF receptor 2 (FGFR2b) 1 . Activation of FGFR2b, either through FGF7 or family members FGF10 and 22 2 , generates a signalling cascade through PI3K/AKT, MAPK and PLCg pathways that upregulate expression of genes such as mTOR and CMYC that function to prevent apoptosis, increase DNA synthesis, accelerate cell cycle progression and other activities important for cell proliferation, differentiation, migration, and survival 3 – 5 . These co-ordinated responses are essential for organ repair and regeneration including of the lung, where FGFR2b is expressed on a variety of airway epithelial cells 6 . This is supported by findings of Ray et al who demonstrated that FGF7 overexpression induced anti-apoptotic signalling by activating AKT, facilitating resistance of murine lung epithelium to oxygen induced injury 7 . The impact of FGFR2 signalling on proliferation and maintenance of the alveolar type II (AT2) cell during lung repair and homeostasis has been extensively reported 4 , 8 – 10 , but temporal signalling is required to allow AT2 differentiation to alveolar type I cells vital for restoration of gas exchange 3 , 11 . However, in chronic or severe airways injury, basal bronchial epithelial cells expressing cytokeratin 5 (KRT5), may contribute to alveolar repair by as much as 50% after bleomycin injury in mice, driven by FGFR2b signalling 8 . The influence of FGF7 on KRT5 + human bronchial epithelial cells (hBECs) is less understood but limited reports suggest an increase in DNA synthesis of primary hBECs 12 , and reduced apoptosis and increased proliferation of the cell-line, 16 Human Bronchial Epithelial (16HBE) cells in response to FGF7 13 . Dysregulated FGFR2b signalling is implicated in the pathogenesis of bronchopulmonary dysplasia 14 , acute lung injury (ALI) 15 , idiopathic pulmonary fibrosis (IPF) 16 and chronic obstructive pulmonary disease (COPD) 17 . In mouse models of pulmonary fibrosis, FGFR2b signalling improved lung repair after bleomycin injury due to enhanced proliferation, migration, and survival of alveolar cells 18 – 21 , and as a result it has been suggested that ligands such as FGF7 may attenuate fibrosis in IPF 22 . Despite the association of FGF7 in airway diseases, supplementation of the growth factor in humans has yielded conflicting results. For example, endogenous FGF7 expression is suppressed during early stages of acute respiratory distress syndrome (ARDS) 15 prompting investigation into the therapeutic potential of FGF7. Recombinant FGF7 (Palifermin) was approved for oral mucositis to prevent epithelial injury during chemotherapy 23 and was investigated for the treatment of ARDS in a human model of lipopolysaccharide (LPS) induced injury which demonstrated increased levels of alveolar marker, surfactant protein D in bronchoalveolar lavage fluid (BALF) 10 . However, in a later clinical trial the protein drug failed to improve the primary outcome of oxygenation index, with worsening outcomes in some patients 24 . The use of recombinant growth factor is often burdened by poor bioavailability, short half-life, systemic toxicity and off-target tissue accumulation including the liver and skin 25 , 26 , driving development of alternative approaches for FGF7 supplementation in the lung. Viral vector overexpression 27 , and bone marrow stem cells engineered to secrete FGF7 28 , have achieved sustained expression of the growth factor however, unregulated overexpression can be detrimental to lung repair. Messenger RNA (mRNA) enables local, transient, and dose-responsive expression of encoded protein and represents an alternative approach for growth factor supplementation in the lung 29 where the potency of FGF7 is greater after local delivery compared to systemic administration 30 , 31 . In addition, unlike Palifermin which is a 23 amino acid N-terminal truncated version of FGF7 produced in bacteria, translation of exogenous mRNA can produce full-length, glycosylated endogenous protein. For personalised medicine, assessment of in vitro response to growth factor therapy using patient derived airway cells that recapitulate disease relevant phenotype is made possible by hBECs accessible from bronchial brushings. However, characterisation of FGF7 influence on these cells is limited, therefore this study first aimed to investigate the impact of recombinant human (rh)FGF7 on primary hBEC proliferation, survival and migration after seeding hBECs from healthy or IPF donors at low density in vitro to promote cell apoptosis 32 . To assess migration, we mechanically injured monolayers of hBECs and measured wound closure in response to FGF7. Furthermore, the production of FGF7 protein from hBECs transfected with modified in vitro transcribed (IVT) mRNA encoding FGF7 was characterised and function of the secreted protein (modFGF7) was investigated in a wound healing. This study demonstrates that exogenous FGF7 increases cell survival and proliferation of hBECs from healthy donors and that mRNA encoded FGF7 may represent a novel therapeutic strategy for enhancing epithelial repair in the injured lung by mediating changes in gene expression that promote migration, proliferation, and survival. However, careful analysis in a personalised and disease relevant context is important to assess patient benefit. METHODS Primary cell culture Primary hBEC and normal human lung fibroblasts (NHLF) from healthy donors (Lonza and Epithelix) or from IPF donors were grown in collagen (Advanced BioMatrix) coated flasks and cultured in Airway Epithelial Growth Medium (Promocell) or Fibroblast Growth Medium-2 (FGM-2) at 37°C and 5% CO 2 . At 70–80% confluence, cells were washed with phosphate buffered saline (PBS) (Gibco) and harvested using TrypLE™ Express Enzyme (Gibco). hBECs were sampled from the airways of patients with IPF undergoing diagnostic bronchoscopy. Fibroblasts were isolated from lung parenchyma of patients with IPF undergoing diagnostic surgical biopsy. Only subjects receiving a multidisciplinary team diagnosis (MDT) of IPF according to current American Thoracic Society (ATS)/European Respiratory Society (ERS)/Japanese Respiratory Society (JRS)/ Latin American Thoracic Association (ALAT) guideline definitions were included (PubMed: 35486072) and the study was approved by the local research ethics committee (15/SC/0101 and 20/SC/0142). Cell proliferation and cytotoxicity assays hBEC were seeded at a density of 2000 cells per 0.32cm 2 in collagen coated 96 well plates (Corning) 24 hours before treatment with PBS (Gibco) or rhFGF7 (Biolegend) between 5–60 pg/µl in PBS. Cell proliferation was monitored for up to 48 hours using the JuLi™ Stage Real-Time Cell History Recorder (NanoTek). Live and dead cells were stained using LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen) according to the manufacturers protocol before imaging at 10x magnification using JuLi™ Stage Real-Time Cell History Recorder. Lactate dehydrogenase (LDH kit, Roche) was quantified relative to positive control cells treated with 1% Triton-X-100 (Sigma) according to manufacturers protocol. Wound healing assay hBEC or NHLF were seeded in precoated PureCol® bovine collagen 0.03 mg/mL (Advanced BioMatrix) 96 well plates the day before transfection with 50 ng FGF7 encoded mRNA (modFGF7), with full 5-methoxyuridine substitution (TriLink, CleanCap AG) complexed with Lipofectamine™ 2000 (Invitrogen) at a mass ratio of 1:4. Supernatant containing secreted modFGF7 protein or media spiked with rhFGF7 (20 pg/µl), was transferred to hBECs every 24 hours for 72 hours before mechanical injury using a pipette tip. Wound closure was monitored over 72 hours using the JuLi™ Stage Real-Time Cell History Recorder (NanoTek) and closure calculated using JuLi™ STAT cell analysis software (v 2.0.0). Secreted modFGF7 protein in supernatant was quantified using a human FGF7 specific enzyme-linked immunosorbent assay kit (Sigma, RAB0188-1KT) according to the manufacturers protocol. Immunofluorescent staining Cells were fixed with 4% paraformaldehyde before permeabilization, blocking, and overnight incubation at 4°C with primary antibodies against Ki67 (Invitrogen, 14569882) or KTR5 (Invitrogen, PA1-37974) or FGF7R2-IIIb (R&D systems, MAB665-SP). After washing, cells were incubated with a species relevant goat IgG conjugated to either Alexa Fluor 488, 555 or 594 (Thermofisher, A-1100) and counterstained with DAPI (Invitrogen). Cells were imaged at 10x magnification using JuLi™. Gene expression studies RNA was isolated using RNeasy Plus Micro Kit (Qiagen) and reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocols. Real-time quantitative PCR (RT-qPCR) was performed using Real Time PCR Systems, ViiA7 (Applied Biosystems, Software Version 1.2.3) or Bio-Rad CFX Opus 384 (CFX Maestro software, version 1.1). Reactions were set up by combining 0.3 µl TaqMan™ gene expression assay (Applied Biosystems), 0.3 µl nuclease-free water, 3 µl TaqMan™ Fast Advanced Master Mix (Applied Biosystems) with 2.4 µl cDNA in a 384-well format. Thermocycling conditions: 50°C/2 min, 95°C/20 sec, 40 cycles of 95°C/1 sec, 60°C/20 sec. Relative expression was calculated using the formula 2 −ΔCT . TaqMan™ gene expression assays used are as follows: ACTB : Hs01060665_g1, AKT1 : Hs00178289_m1, CMYC : Hs00153408_m1, FGFR2 : Hs00256382_m1, MAPK1 : Hs01046830_m1, MAPK3 : Hs00385075_m1, MTOR : Hs00234508_m1, PIK3CA : Hs00180679_m1, PLCG1 : Hs06639798_s1. Statistics Statistical analyses were performed in GraphPad Prism 10 (Version 10.3.1 (464)). Statistical analyses are indicated in the figure legends and significance between groups was assessed with a significance level of P < 0.05. Data from 2–6 primary cell donors, with 2–3 technical replicates each, are shown as mean and standard deviation unless otherwise indicated. RESULTS rhFGF7 increases bronchial epithelial cell proliferation under physiological stress When seeded at low density, anchorage dependent epithelial cells can undergo anoikis due to loss of contact to neighbouring cells, preventing growth and proliferation. This phenomenon was observed in control KRT5 + hBECs, where sub-optimal seeding density resulted in a lack of increased confluency up to 40 hours post-seeding (11.7%±2.7%) (Fig. 1 A). In contrast, hBECs treated with 5–30 pg/µl of rhFGF7 demonstrated a dose-dependent increase in cell confluency, which was significant at all doses (5: P < 0.05, 10: P < 0.01, 20: P < 0.001, 30: P < 0.0001), ranging from 19.6 ± 6.8% at the 5 pg/µl dose, up to 25.6 ± 3.0%, at 30 pg/µl. Although the 60 pg/µl dose significantly increased hBEC confluency compared to baseline (19.9 ± 4.6%, P < 0.01), this was lower than that of the 30 pg/µl rhFGF7 dose suggesting lower doses are sufficient to stimulate hBEC proliferation (Fig. 1 B). The proportion of proliferating cells was quantified by immunofluorescent staining of Ki67 40 hours post-treatment. Cells treated with 5 pg/µl dose of rhFGF7 demonstrated a non-significant increase in Ki67 positive cells (54.0 ± 11.7%) compared to PBS control (37.2 ± 23.0%). Treatment with higher doses of 10–60 pg/µl rhFGF7 facilitated a significant increase in the proportion of actively proliferating, Ki67 positive cells (57.1 ± 8.8% to 63.4 ± 9.6%; 10–30 pg/µl: P < 0.05, 60 pg/µl: P < 0.01) (Fig. 1 C, D). rhFGF7 improves survival of bronchial epithelial cells seeded at low density. In agreement with the confluency assay, the physiological stressor of low seeding density promoted hBEC apoptosis as indicated by lactate dehydrogenase (LDH) release, with control cells secreting 115.7 ± 11.7% LDH at 40 hours post-seeding relative to Triton X (Fig. 2 A). When hBECs were treated with 10–60 pg/µl rhFGF7, a significant reduction in cytotoxicity was observed (47.7 ± 8.3% − 40.6 ± 10.6%, P < 0.001). Cells treated with the lowest dose of 5 pg/µl rhFGF7 did not express reduced cytotoxicity, with LDH release of 103.3 ± 56.9% (Fig. 2 A). Together, these data suggest a range of 10–30 pg/µl rhFGF7 may be protective against primary hBEC cell death in vitro . A dose of 20 pg/µl was selected for all future experiments, we first confirmed that this dose would increase the proportion of live cells while reducing dead cells as expected based on previous results. The proportion of live cells following treatment with 20 pg/µl rhFGF7 was significantly higher (73.4 ± 5.2%, P < 0.0001) than that of control cells, where the majority were dead at the 40 hours post-seeding and live cell number was reduced to 19.1 ± 3.2% (Fig. 2 B). After confirming that FGF7 can impact hBEC proliferation and survival, next it was investigated whether synthetic mRNA encoding FGF7 could be translated in hBECs and impact wound closure compared to rhFGF7. Prophylactic application of recombinant or mRNA derived FGF7 to hBEC monolayers improves repair. Epithelial cells do not endogenously express FGF7, however when transfected with exogenous FGF7 mRNA, primary hBECs are capable of significant dose-dependent FGF7 translation and secretion (50 ng mRNA: 55.44 ± 32.06 pg, P < 0.05; 100 ng mRNA: 157.90 ± 43.14 pg, P < 0.0001) compared to non-transfected hBECs (0.86 ± 0.33 pg) (Fig. 3 A). Translation of FGF7 mRNA was transient with FGF7 protein levels peaking at 12–24 hours post-transfection (Fig. 3 B). To determine whether FGF7 influences hBEC wound repair, a prophylactic strategy was implemented as previously described 33 . Primary hBECs were treated with FGF7 secreted from mRNA transfected hBECs (modFGF7) or rhFGF7 and wounded 72 hours post-treatment. Wounded hBECs treated with modFGF7 (36.1 ± 21.1%, P < 0.05) or rhFGF7 (50.5 ± 21.5%, P < 0.001) both demonstrated significantly increased wound closure at 36-hours post-injury compared to PBS treated wounds (15.3 ± 13.3%) (Fig. 3 C). At 72-hours post-injury, treated cells achieved wound closure while control wounds remained unhealed (Fig. 3 D), possibly due to the additional contribution of increased survival and proliferation at the longer timepoint. The improvement in wound closure in hBECs that received FGF7 was accompanied by upregulation in genes that are key to re-epithelialisation during repair by mediation of genes associated with cell survival, growth, proliferation, and migration (RAC1, CDH1, CTNNB1, PI3KCA, MAPK1/3, MTOR , and CMYC). Interestingly, FGFR2 and PLCG1 were also upregulated in FGF7 treated and healed wounds suggesting activation of signal transduction through the FGF7 receptor (Fig. 3 E). Greater wound repair was observed in rhFGF7 treated wounds compared to modFGF7, and similarly higher expression of endogenous epithelial repair genes was observed with rhFGF7 treated cells (Fig. 3 C, E). IPF hBECs demonstrate active survival, proliferation, and migration that is not impacted by FGF7 treatment . After establishing positive responses to FGF7 in normal or ‘healthy’ hBECs, we sought to investigate how hBECs from fibrotic lung would respond to the growth factor. First, it was confirmed that hBECs from IPF donors could translate FGF7 mRNA. Cells were transfected at two dose levels and resulted in significant and dose-dependent secretion of FGF protein as measured by ELISA over 72-hours (50ng: 31.44 ± 8.36 pg, P < 0.0001; 100 ng: 60.16 ± 12.80 pg, P < 0.0001) compared to non-transfected cells (0.14 ± 0.19 pg) (Fig. 4 A). In addition, transient expression kinetics followed a similar pattern to healthy hBECS, with peak FGF7 detected between 12–24 hours post-transfection (Fig. 4 B). To determine if prophylactic FGF7 treatment could impact IPF hBEC migration in a wound closure assay, supernatant from FGF7 mRNA transfected cells or media spiked with rhFGF7 was applied to IPF hBECs prior to mechanical injury. Control, PBS treated IPF hBEC wounds were capable of notable wound closure 36 hours post-wounding (27.1 ± 9.7%) (Fig. 4 C) and treatment with FGF7 did not improve closure (rhFGF7: 28.6 ± 19.5% and modFGF7: 16.2 ± 13.2%) (Fig. 4 C). Relative expression of key epithelial repair genes involved in proliferation, survival and migration were found to be largely unchanged in FGF7 treated wounds compared to PBS control (Fig. 4 D). In a low seeding density assay, IPF-hBECs almost doubled in confluency in both rhFGF7 treated and PBS control conditions (20.9%8.1 ± and 18.8%±9.6, respectively) 40 hours post-seeding (Fig. 4 E). Cytotoxicity was measured by LDH release and confirmed that IPF hBECs were relatively resistant to cell death compared to healthy hBECs with only 27.4% (± 13.0) LDH release observed in PBS control and 23.9% (± 7.4) in rhFGF7 treated conditions (Fig. 4 F). To further explore the notable rates of proliferation and repair in IPF hBEC, and limited response to FGF7, baseline gene expression of hBECs from IPF and healthy donors cultured under standard conditions was compared. FGFR2 receptor gene expression was similar both in healthy and IPF hBECs confirmed by PCR and protein expression confirmed by immunostaining for FGFR2b against KRT5 (Fig. 5 A). However, gene expression downstream of the FGF7 signalling axis were elevated in IPF hBECs compared to healthy controls (Fig. 5 B). Expression of genes related to cell proliferation and pro-survival was significantly upregulated; PLCG1 (P < 0.05), MTOR (P < 0.05), PIK3CA (P < 0.01), and AKT (P < 0.01). Expression of CMYC, MAPK1 , and MAPK3 was higher in IPF hBECs than in healthy controls, although not significant (Fig. 5 B). Expression of TGFB1 , a potent suppressor of endogenous FGF7 production 34 , was significantly higher in IPF hBECs (P < 0.01). DISCUSSION Denudation and apoptosis of lung epithelium can occur during acute or chronic lung injury and dysfunctional repair is implicated in the pathogenesis of pulmonary diseases such as IPF 16 , 35 and COPD 17 . To investigate the impact of the epithelial mitogen FGF7 on hBECs in vitro , low seeding density was employed as a physiological stressor to reduce cell-cell interaction, which inhibits cell growth and proliferation, ultimately leading to programmed cell death 32 . We hypothesised that rhFGF7 treatment of primary hBEC would enhance proliferation and survival under these conditions. In this study, rhFGF7 treatment enhanced healthy hBEC survival at low seeding densities. Cell proliferation was enhanced 2-fold compared to PBS control treated cells while cytotoxicity was reduced by 71%. Moreover, rhFGF7 treatment enhanced the proportion of live cells compared to PBS control treated cells. Balasooriya et al report similar findings of FGF7’s ability to increase proliferation and growth of low-density murine airway basal cells compared to untreated cells 11 . In addition, Shyamsundar et al utilised bronchoalveolar lavage (BAL) fluid from Palifermin treated subjects with LPS induced acute lung injury to treat alveolar epithelial cells in vitro and demonstrated significant improvements in cell proliferation of the A549 adenocarcinoma cell line. However, the level of FGF7 in the BAL fluid of treated and untreated subjects was not quantified 10 . Localised and transient protein therapy is desirable when systemic toxicity is dose-limiting but where this is difficult to achieve with recombinant proteins, modified IVT mRNA may be an alternative modality for protein replacement. Primary hBECs do not endogenously express FGF7, therefore we established whether FGF7 encoded mRNA could be translated to functional protein by hBECs. For the first time, we demonstrate non-viral delivery of modified FGF7 mRNA generates dose dependent levels of FGF7 protein secreted from primary hBECs. However, variability in protein levels produced by different donors was observed which may impact therapeutic dosing of IVT mRNA. In addition, we transfected primary normal human lung fibroblasts (NHLF) with FGF7 mRNA and measured higher levels of secreted FGF7 post-transfection (Fig. S1) possibly due to NHLF being innate producers of this growth factor, and therefore more efficient at FGF7 translation and processing 36 . As well as FGF7, fibroblasts secrete various mediators that augment the epithelial repair process such as interleukin 6 (IL-6) 37 , interleukin 1β (IL-1β) 38 , and epidermal growth factor (EGF) 39 and dermal fibroblast conditioned media alone has been shown to enhance keratinocyte wound repair 40 , therefore our study utilised supernatant of transfected hBEC for assessing the impact of modFGF7 on hBEC wound closure. This work represents the first demonstration that pre-treatment of primary hBECs with rhFGF7 or modFGF7 secreted from mRNA transfected hBECs, significantly improves wound closure following mechanical injury. Wang et al recently reported FGF7 treatment improves wound closure and proliferation of mechanically injured 16HBE cells 13 and Denzinger et al reported similar findings with supernatants from FGF7-mRNA transfected keratinocytes improving wound closure of scratched keratinocyte monolayers 33 . The overall improvement in healthy hBEC wound closure was accompanied by upregulation in genes associated with epithelial repair and FGF7 signal transduction. Activation and dimerization of FGFR2b is demonstrated by upregulation in PLCG1 , encoding adaptor protein phospholipase C-γ (PLCγ) which is recruited to the activated receptor to facilitate recruitment of downstream signalling molecules 25 . Increased expression of RAC1 , a key regulator of the cell cycle, cell-cell adhesion, and cell motility 41 as well as key genes involved in re-epithelisation and cell-cell adhesion, CDH1 , encoding E-cadherin, and CTNNB1 , encoding β-catenin, was observed in wounds with improved healing 42 . Activation of the PI3K/AKT and MAPK pathways in FGF7 treated hBEC wounds was demonstrated by upregulation in PIK3CA , MAPK1 , and MAPK3. Interestingly, AKT1 and MTOR upregulation was observed in rhFGF7 but not modFGF7 treated wounds potentially due to lack of quantification of modFGF7 protein level in the supernatants of mRNA transfected cells, preventing matched dosing. Upregulation of MAPK1, MAPK3 , and CMYC expression was observed in both rhFGF7 and modFGF7 treated wounds at 72 hours post-injury thereby promoting cell survival and proliferation. Muyal et al report similar findings of exogenous FGF7 mediated activation of signalling through the MAPK pathway in emphysematous mice with upregulated MAPK1 and MAPK3 expression. In addition, increased expression of CMYC was also demonstrated in this study 43 . Interestingly, in response to FGF7 treatment, upregulation of FGFR2 was observed in healthy hBECs. Together, these data suggest improved primary hBEC wound repair in response to prophylactic FGF7 is mediated by activation of genes that promote cell survival, proliferation, and migration. Intracellular signal transduction is complex, with many genes involved in the regulation of cell fate and behaviour, thus we acknowledge that this study only interrogates a small panel of genes responsible for epithelial repair. Furthermore, we only assayed changes in gene expression at the endpoint of the injury assay therefore temporal changes in expression were not captured. Bronchial epithelial cells may contribute to fibrotic lung disease through various mechanisms including ectopic expansion of KRT5 + BECs in distal lung 35 , 44 , and overexpression of transforming growth factor β1 (TGF-β1) which is a potent inducer of fibrosis and negative regulator of endogenous FGF7 production 34 . A reduction in FGFR2 expression in regions undergoing remodelling in the IPF lung has also been observed 45 . We found no difference in baseline FGFR2 expression between healthy and IPF hBECs but did observe increased gene expression of TGF-β1 . When cells were seeded at low density, control IPF hBECs increased in confluency to a greater extent than control healthy hBECs and treatment with rhFGF7 did not impact proliferation of IPF hBECs. Moreover, wound closure of untreated IPF hBEC wounds was higher than that of healthy hBEC control wounds, and prophylactic treatment of IPF hBECs with FGF7 did not improve wound closure. Unlike healthy hBECs, expression of genes related to survival, proliferation and migration were unchanged between FGF7 treated and PBS control IPF hBECs, although a trend in upregulation of FGFR2 and TP63 was observed in some donors (Fig. S2). Due to limitations in IPF cell numbers, increasing doses of FGF7 could not be tested on IPF-HBECs which may require higher doses to elicit a response. Finally, we acknowledge that submerged, monolayer culture of IPF hBECs is not representative of the spatial and temporal heterogeneity of IPF where areas of early and established fibrosis are present 46 . Nevertheless, it has previously been reported that hBECs 47 and fibroblasts from IPF lungs 48 have increased baseline pAKT/PI3K signalling leading to increased cell proliferation and survival, consequently inhibition of this pathway in fibroblasts is a pharmacological target to reduce fibrosis 49 . However, IPF hBECs also undergo apoptosis after inhibition of PI3K/mTOR 47 , and off-target systemic epithelial cytotoxicity due to non-specific inhibition remains unknown. Consequently, there may be rationale for further exploration of FGF7 as a protective growth factor during pharmacological interventions that induce epithelial apoptosis such as non-selective PI3K/mTOR inhibition or bleomycin chemotherapy. This study demonstrates the feasibility of exogenous FGF7 encoded mRNA as a novel therapeutic strategy for repair of normal primary bronchial epithelial cells. Moreover, we demonstrate that exogenous FGF7 can induce FGFR2 gene expression and activate intracellular signalling that promotes primary hBEC proliferation, migration, and survival. Application to other diseases where impaired epithelial repair is caused by dysregulated FGFR2b signal transduction should be investigated. Statements & Declarations Funding: The authors would like to acknowledge funding from the UKRI Advanced Therapies Network (ref: CiC013), Medical Research Council (MR/W028433/1) and the Rosetrees Trust (ref: PhD2022\100016). The work was supported by the National Institute for Health & Care Research through the Imperial Biomedical Research Centre. Competing Interests: The authors have no relevant financial or non-financial competing interests to disclose. Author Contributions: Asha Patel, Philip Molyneaux and Skye Quinn contributed to the study conception and design. Material preparation, data collection and analysis were performed by Skye Quinn, Asha Patel, Alexander Jenkins and Rafaela Konstantinidi. The first draft of the manuscript was written by Skye Quinn and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability: The datasets generated during the current study are available from the corresponding author on reasonable request. Ethics approval: Written informed consent was obtained from all subjects, and the study was approved by the local Research Ethics Committee (15/SC/0101 and 20/SC/0142). Clinical trial number: not applicable. References Miki, T. et al. Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene. Proc Natl Acad Sci U S A 89 , (1992). Zhang, X. et al. Receptor Specificity of the Fibroblast Growth Factor Family. Journal of Biological Chemistry 281 , (2006). Liberti, D. 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D., Lucas, J. J. & Mason, R. J. Transforming growth factor-β antagonizes alveolar type II cell proliferation induced by keratinocyte growth factor. Am J Respir Cell Mol Biol 31 , 679–686 (2004). Galiacy, S. et al. Keratinocyte growth factor promotes cell motility during alveolar epithelial repair in vitro. Exp Cell Res 283 , 215–229 (2003). Ulich, T. R. et al. Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. Journal of Clinical Investigation 93 , 1298–1306 (1994). Dorry, S. J., Ansbro, B. O., Ornitz, D. M., Mutlu, G. M. & Guzy, R. D. FGFR2 Is Required for AEC2 Homeostasis and Survival after Bleomycin-induced Lung Injury. Am J Respir Cell Mol Biol 62 , 608–621 (2019). Spielberger, R. et al. Palifermin for Oral Mucositis after Intensive Therapy for Hematologic Cancers. New England Journal of Medicine 351 , 2590–2598 (2004). McAuley, D. F. et al. Keratinocyte growth factor for the treatment of the acute respiratory distress syndrome (KARE): a randomised, double-blind, placebo-controlled phase 2 trial. Lancet Respir Med 5 , 484–491 (2017). Sadeghi, S. et al. Keratinocyte growth factor in focus: A comprehensive review from structural and functional aspects to therapeutic applications of palifermin. Int J Biol Macromol 191 , (2021). Zia-Amirhosseini, P. et al. Pharmacokinetics, pharmacodynamics, and safety assessment of palifermin (rHuKGF) in healthy volunteers. Clin Pharmacol Ther 79 , (2006). Sakamoto, S. et al. Keratinocyte growth factor gene transduction ameliorates pulmonary fibrosis induced by bleomycin in mice. Am J Respir Cell Mol Biol 45 , (2011). Aguilar, S. et al. Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis. PLoS One 4 , (2009). Patel, A. K. et al. Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium. Advanced Materials 31 , 1805116 (2019). Ulich, T. R. et al. Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. Journal of Clinical Investigation 93 , (1994). Guo, J. et al. Intravenous keratinocyte growth factor protects against experimental pulmonary injury. Am J Physiol Lung Cell Mol Physiol 275 , (1998). Frisch, S. M. & Francis, H. Disruption of epithelial cell-matrix interactions induces apoptosis. Journal of Cell Biology 124 , 619–626 (1994). Denzinger, M. et al. Keratinocyte growth factor modified messenger RNA accelerating cell proliferation and migration of keratinocytes. Nucleic Acid Ther 28 , (2018). Correll, K. A. et al. TGF beta inhibits HGF, FGF7, and FGF10 expression in normal and IPF lung fibroblasts. Physiol Rep 6 , (2018). Hewitt, R. J. et al. Lung extracellular matrix modulates KRT5+ basal cell activity in pulmonary fibrosis. Nat Commun 14 , (2023). Rubin, J. S. et al. Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc Natl Acad Sci U S A 86 , (1989). Voiriot, G. et al. Interleukin-6 displays lung anti-inflammatory properties and exerts protective hemodynamic effects in a double-hit murine acute lung injury. Respir Res 18 , (2017). Geiser, T., Jarreau, P. H., Atabai, K. & Matthay, M. A. Interleukin-1β augments in vitro alveolar epithelial repair. Am J Physiol Lung Cell Mol Physiol 279 , (2000). Finigan, J. H., Downey, G. P. & Kern, J. A. Human epidermal growth factor receptor signaling in acute lung injury. American Journal of Respiratory Cell and Molecular Biology vol. 47 Preprint at https://doi.org/10.1165/rcmb.2012-0100TR (2012). Maarof, M., Chowdhury, S. R., Saim, A., Idrus, R. B. H. & Lokanathan, Y. Concentration dependent effect of human dermal fibroblast conditioned medium (Dfcm) from three various origins on keratinocytes wound healing. Int J Mol Sci 21 , (2020). Desai, L. P., Aryal, A. M., Ceacareanu, B., Hassid, A. & Waters, C. M. RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 287 , (2004). Leoni, G., Neumann, P. A., Sumagin, R., Denning, T. L. & Nusrat, A. Wound repair: Role of immune-epithelial interactions. Mucosal Immunology vol. 8 Preprint at https://doi.org/10.1038/mi.2015.63 (2015). Muyal, J. P., Kotnala, S., Bhardwaj, H. & Tyagi, A. Effect of recombinant human keratinocyte growth factor in inducing Ras-Raf-Erk pathway-mediated cell proliferation in emphysematous mice lung. Inhal Toxicol 26 , 761–771 (2014). Plantier, L. et al. Ectopic respiratory epithelial cell differentiation in bronchiolised distal airspaces in idiopathic pulmonary fibrosis. Thorax 66 , (2011). El Agha, E. et al. Is the fibroblast growth factor signaling pathway a victim of receptor tyrosine kinase inhibition in pulmonary parenchymal and vascular remodeling? Am J Physiol Lung Cell Mol Physiol 315 , L248–L252 (2018). Plantier, L. et al. Physiology of the lung in idiopathic pulmonary fibrosis. Eur Respir Rev 27 , (2018). Mercer, P. F. et al. Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF. Thorax 71 , (2016). Xia, H. et al. Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis. Journal of Experimental Medicine 205 , (2008). Lukey, P. T. et al. A randomised, placebo-controlled study of omipalisib (PI3K/mTOR) in idiopathic pulmonary fibrosis. European Respiratory Journal 53 , (2019). Supplementary Files Supplementaryfigures.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 08 Jul, 2025 Editor assigned by journal 24 Jun, 2025 First submitted to journal 23 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6939652","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482300898,"identity":"ac958d15-d961-45cb-9513-0ee3bce827c6","order_by":0,"name":"Skye Quinn","email":"","orcid":"","institution":"Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Skye","middleName":"","lastName":"Quinn","suffix":""},{"id":482300899,"identity":"b5cf51c3-1ca9-4f37-a672-d011cb5fd922","order_by":1,"name":"Alexander Jenkins","email":"","orcid":"","institution":"Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Jenkins","suffix":""},{"id":482300900,"identity":"2f7da887-b3d9-4f51-ae68-85f6ca07fc1c","order_by":2,"name":"Rafaela Konstantinidi","email":"","orcid":"","institution":"Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Rafaela","middleName":"","lastName":"Konstantinidi","suffix":""},{"id":482300901,"identity":"fb1d70a6-8d92-429e-8615-ed6aa64cbbb0","order_by":3,"name":"Philip L Molyneaux","email":"","orcid":"","institution":"Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Philip","middleName":"L","lastName":"Molyneaux","suffix":""},{"id":482300902,"identity":"d767a6e5-3d31-4c52-8e89-f613a695823a","order_by":4,"name":"Asha Kumari Patel","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-7266-9251","institution":"Imperial College London","correspondingAuthor":true,"prefix":"","firstName":"Asha","middleName":"Kumari","lastName":"Patel","suffix":""}],"badges":[],"createdAt":"2025-06-20 14:15:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6939652/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6939652/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86437371,"identity":"d4a61259-ecf8-4345-94f2-5f906b22a763","added_by":"auto","created_at":"2025-07-10 15:46:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":178604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfluence of rhFGF7 on proliferation of primary hBEC seeded at low confluency. \u003c/strong\u003e(A, B) Confluency (%) of primary human bronchial epithelial cells 40 hours after treatment with 0-60 pg/µl rhFGF7 (Two-way ANOVA with Sidaks multiple comparison). (C) Ki67 positive cells (%) at 40-hour endpoint (One-way ANOVA with Dunnetts multiple comparison). (D) Representative images depicting DAPI (blue), KRT5 (green), Ki67 (magenta) immunofluorescent staining. The first panel is brightfield and i67 merged images. Scale bars = 100 µm. n = 2 donors with 3 replicates each. *P\u0026lt;0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6939652/v1/ab74497e12bf4ae14e3f9a23.png"},{"id":86437372,"identity":"8cfaa1e6-042e-435e-a1ea-272afe2b9088","added_by":"auto","created_at":"2025-07-10 15:46:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":157100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eInfluence of rhFGF7 on primary hBEC survival when seeded at low density. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;(A) Primary human bronchial epithelial cell (hBEC) lactate dehydrogenase (LDH) release (%) at 40-hours post-treatment with 0-60 pg/µl rhFGF7 relative to Triton X control (dotted line). One-way ANOVA with Dunnetts multiple comparison, n = 2 primary cell donors, 3 technical replicates. (B) Proportion of live versus dead cells at 40-hours post-treatment with 20 pg/µl rhFGF or placebo control. Two-way ANOVA with Sidaks multiple comparison, n = 3 technical replicates. (C) Representative immunofluorescent imaging depicting live and dead cells in control versus rhFGF7 treated hBECs. Scale bars = 200 µm. \u003c/em\u003e***P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6939652/v1/fc2b122b803bd02c7f9bbb46.png"},{"id":86437376,"identity":"947ebdf6-9d3b-4b5f-abc4-0ed018c933f5","added_by":"auto","created_at":"2025-07-10 15:46:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":278756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranslation of FGF7 mRNA in healthy primary hBECs and impact of FGF7 on wound closure. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003ePrimary human bronchial epithelial cells were transfected with 50 or 100 ng of FGF7 mRNA and secreted cumulative FGF7 protein from the supernatant of primary hBECs was measured over 72 hours and (B) measured at multiple time points to capture expression kinetics (n = 2 primary donors). (C) Recombinant FGF7 protein (20 pg/mL rhFGF7) or supernatant from hBECs transfected with 50 ng FGF7 mRNA \u0026nbsp;(modFGF7) was applied to wounded hBECs. Closure was measured at 36 hours and compared to media only control (n = 3 replicates in 4 primary donors). (D) Representative brightfield images of hBEC wounds at final end point of 72 h (scale = 250 µm). (E) Heat map of log2 fold change (Log2FC) in gene expression relative to ACTB housekeeping gene and normalised to media only control at wound closure end point (n=4 donors). One-way ANOVA with Dunnetts multiple comparison. *P\u0026lt;0.05, ***P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6939652/v1/af27de86e3908acb3759ca54.png"},{"id":86438140,"identity":"e852d463-98ca-43aa-be8b-b0d159757557","added_by":"auto","created_at":"2025-07-10 15:54:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":137282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranslation of FGF7 mRNA in primary hBECs from donors with Idiopathic Pulmonary Fibrosis and impact of FGF7 on wound closure and confluency. \u003c/strong\u003e(A) IPF\u003cstrong\u003e-\u003c/strong\u003ehBECs were transfected with varying doses of FGF7 mRNA and secreted FGF7 protein measured over 72 hours (One-way ANOVA with Dunnetts multiple comparison) and (B) measured at multiple time points to capture expression kinetics (n = 3 donors, 3 technical repeats). (C) Recombinant FGF7 protein (20 pg/mL rhFGF7) or supernatant from hBECs transfected with 50 ng FGF7 mRNA (modFGF7) was applied prophylactically to IPF-hBECs before mechanical injury. Wound closure (%) was measured 36 hours after injury and compared to media only control (One-way ANOVA with Dunnetts multiple comparison; n = 3 primary donors). (D) Heat map of log2 fold change in gene expression relative to ACTB gene and normalised to media only control at wound closure end point. (E) Impact of rhFGF7 on IPF-hBEC confluency when seeded at low density, compared to vehicle control and (F) levels of lactate dehydrogenase measured at 40 hours post-seeding. (n=3 primary donors, unpaired t test).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6939652/v1/e55619b73883f7f54449a561.png"},{"id":86438557,"identity":"eee1fd52-c38d-4c83-8428-dc0994e9ffd5","added_by":"auto","created_at":"2025-07-10 16:02:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":253191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFGFR2 receptor expression and relative gene expression in healthy vs diseased (IPF) bronchial epithelial cells\u003c/strong\u003e \u003cstrong\u003eunder standard cultivation conditions.\u003c/strong\u003e (A) Representative images depicting DAPI (blue), KRT5 (yellow), FGFR2b (magenta) immunofluorescent staining of healthy and IPF-hBECs. Scale bars = 32 µm and baseline FGFR2 receptor gene expression in hBECs relative to ACTB endogenous control gene (****P\u0026lt;0.0001, Unpaired t test; n = 5 IPF and 6 healthy donors). Baseline expression of genes related to proliferation, migration, survival, and repair in non-wounded and untreated primary hBECs from donors with IPF (n= 5 primary donors) or healthy control (n= 4 primary donors,). Unpaired t test. *P\u0026lt;0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6939652/v1/880a60919a910190c6d76592.png"},{"id":86439986,"identity":"75cad541-b977-48e2-b70b-28aa1e4ea46e","added_by":"auto","created_at":"2025-07-10 16:18:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1824080,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6939652/v1/9563a126-4061-45c1-95cd-40937e5bf77b.pdf"},{"id":86438138,"identity":"5fb33851-34e3-445e-8f4d-bd016d583fc8","added_by":"auto","created_at":"2025-07-10 15:54:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1079864,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6939652/v1/050d81d131fd6c925ebbcb51.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eFibroblast growth factor-7 promotes repair of primary human bronchial epithelial cells\u003c/p\u003e","fulltext":[{"header":"Key Messages","content":"\u003cul\u003e\n \u003cli\u003eFGF7 is an epithelial mitogen previously shown to improve repair and regeneration of oral mucosa, keratinocytes and alveolar cells. This study explores impact on human bronchial epithelial cells (hBEC).\u003c/li\u003e\n \u003cli\u003eFGF7 increases proliferation, survival and migration of primary hBEC.\u003c/li\u003e\n \u003cli\u003emRNA encoding FGF7 provides an alternative approach to growth factor supplementation.\u003c/li\u003e\n \u003cli\u003ePost-FGF7 treatment, genes associated with FGF7 receptor activation and epithelial repair are upregulated in healthy hBECs but not hBECs from fibrotic lung due to higher baseline levels of these genes in untreated IPF-hBECs.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eFibroblast growth factor 7 (FGF7 or Keratinocyte growth factor; KGF) is a potent mitogen secreted by mesenchymal cells such as fibroblasts, and specifically targets epithelial cells by binding to its single known receptor, the IIIb isoform of FGF receptor 2 (FGFR2b)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Activation of FGFR2b, either through FGF7 or family members FGF10 and 22\u003csup\u003e2\u003c/sup\u003e, generates a signalling cascade through PI3K/AKT, MAPK and PLCg pathways that upregulate expression of genes such as \u003cem\u003emTOR\u003c/em\u003e and \u003cem\u003eCMYC\u003c/em\u003e that function to prevent apoptosis, increase DNA synthesis, accelerate cell cycle progression and other activities important for cell proliferation, differentiation, migration, and survival \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These co-ordinated responses are essential for organ repair and regeneration including of the lung, where FGFR2b is expressed on a variety of airway epithelial cells\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This is supported by findings of Ray \u003cem\u003eet al\u003c/em\u003e who demonstrated that FGF7 overexpression induced anti-apoptotic signalling by activating AKT, facilitating resistance of murine lung epithelium to oxygen induced injury\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The impact of FGFR2 signalling on proliferation and maintenance of the alveolar type II (AT2) cell during lung repair and homeostasis has been extensively reported\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, but temporal signalling is required to allow AT2 differentiation to alveolar type I cells vital for restoration of gas exchange\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, in chronic or severe airways injury, basal bronchial epithelial cells expressing cytokeratin 5 (KRT5), may contribute to alveolar repair by as much as 50% after bleomycin injury in mice, driven by FGFR2b signalling\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The influence of FGF7 on KRT5\u0026thinsp;+\u0026thinsp;human bronchial epithelial cells (hBECs) is less understood but limited reports suggest an increase in DNA synthesis of primary hBECs\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and reduced apoptosis and increased proliferation of the cell-line, 16 Human Bronchial Epithelial (16HBE) cells in response to FGF7\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDysregulated FGFR2b signalling is implicated in the pathogenesis of bronchopulmonary dysplasia\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, acute lung injury (ALI)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, idiopathic pulmonary fibrosis (IPF)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and chronic obstructive pulmonary disease (COPD)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In mouse models of pulmonary fibrosis, FGFR2b signalling improved lung repair after bleomycin injury due to enhanced proliferation, migration, and survival of alveolar cells\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and as a result it has been suggested that ligands such as FGF7 may attenuate fibrosis in IPF\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Despite the association of FGF7 in airway diseases, supplementation of the growth factor in humans has yielded conflicting results. For example, endogenous FGF7 expression is suppressed during early stages of acute respiratory distress syndrome (ARDS)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e prompting investigation into the therapeutic potential of FGF7. Recombinant FGF7 (Palifermin) was approved for oral mucositis to prevent epithelial injury during chemotherapy\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and was investigated for the treatment of ARDS in a human model of lipopolysaccharide (LPS) induced injury which demonstrated increased levels of alveolar marker, surfactant protein D in bronchoalveolar lavage fluid (BALF)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, in a later clinical trial the protein drug failed to improve the primary outcome of oxygenation index, with worsening outcomes in some patients\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe use of recombinant growth factor is often burdened by poor bioavailability, short half-life, systemic toxicity and off-target tissue accumulation including the liver and skin\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, driving development of alternative approaches for FGF7 supplementation in the lung. Viral vector overexpression\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and bone marrow stem cells engineered to secrete FGF7\u003csup\u003e28\u003c/sup\u003e, have achieved sustained expression of the growth factor however, unregulated overexpression can be detrimental to lung repair. Messenger RNA (mRNA) enables local, transient, and dose-responsive expression of encoded protein and represents an alternative approach for growth factor supplementation in the lung\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e where the potency of FGF7 is greater after local delivery compared to systemic administration\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In addition, unlike Palifermin which is a 23 amino acid N-terminal truncated version of FGF7 produced in bacteria, translation of exogenous mRNA can produce full-length, glycosylated endogenous protein.\u003c/p\u003e\u003cp\u003eFor personalised medicine, assessment of \u003cem\u003ein vitro\u003c/em\u003e response to growth factor therapy using patient derived airway cells that recapitulate disease relevant phenotype is made possible by hBECs accessible from bronchial brushings. However, characterisation of FGF7 influence on these cells is limited, therefore this study first aimed to investigate the impact of recombinant human (rh)FGF7 on primary hBEC proliferation, survival and migration after seeding hBECs from healthy or IPF donors at low density \u003cem\u003ein vitro\u003c/em\u003e to promote cell apoptosis\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To assess migration, we mechanically injured monolayers of hBECs and measured wound closure in response to FGF7. Furthermore, the production of FGF7 protein from hBECs transfected with modified \u003cem\u003ein vitro\u003c/em\u003e transcribed (IVT) mRNA encoding FGF7 was characterised and function of the secreted protein (modFGF7) was investigated in a wound healing. This study demonstrates that exogenous FGF7 increases cell survival and proliferation of hBECs from healthy donors and that mRNA encoded FGF7 may represent a novel therapeutic strategy for enhancing epithelial repair in the injured lung by mediating changes in gene expression that promote migration, proliferation, and survival. However, careful analysis in a personalised and disease relevant context is important to assess patient benefit.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePrimary cell culture\u003c/h2\u003e\u003cp\u003ePrimary hBEC and normal human lung fibroblasts (NHLF) from healthy donors (Lonza and Epithelix) or from IPF donors were grown in collagen (Advanced BioMatrix) coated flasks and cultured in Airway Epithelial Growth Medium (Promocell) or Fibroblast Growth Medium-2 (FGM-2) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. At 70\u0026ndash;80% confluence, cells were washed with phosphate buffered saline (PBS) (Gibco) and harvested using TrypLE\u0026trade; Express Enzyme (Gibco).\u003c/p\u003e\u003cp\u003ehBECs were sampled from the airways of patients with IPF undergoing diagnostic bronchoscopy. Fibroblasts were isolated from lung parenchyma of patients with IPF undergoing diagnostic surgical biopsy. Only subjects receiving a multidisciplinary team diagnosis (MDT) of IPF according to current American Thoracic Society (ATS)/European Respiratory Society (ERS)/Japanese Respiratory Society (JRS)/ Latin American Thoracic Association (ALAT) guideline definitions were included (PubMed: 35486072) and the study was approved by the local research ethics committee (15/SC/0101 and 20/SC/0142).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell proliferation and cytotoxicity assays\u003c/h3\u003e\n\u003cp\u003ehBEC were seeded at a density of 2000 cells per 0.32cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e in collagen coated 96 well plates (Corning) 24 hours before treatment with PBS (Gibco) or rhFGF7 (Biolegend) between 5\u0026ndash;60 pg/\u0026micro;l in PBS. Cell proliferation was monitored for up to 48 hours using the JuLi\u0026trade; Stage Real-Time Cell History Recorder (NanoTek). Live and dead cells were stained using LIVE/DEAD\u0026trade; Viability/Cytotoxicity Kit (Invitrogen) according to the manufacturers protocol before imaging at 10x magnification using JuLi\u0026trade; Stage Real-Time Cell History Recorder. Lactate dehydrogenase (LDH kit, Roche) was quantified relative to positive control cells treated with 1% Triton-X-100 (Sigma) according to manufacturers protocol.\u003c/p\u003e\n\u003ch3\u003eWound healing assay\u003c/h3\u003e\n\u003cp\u003ehBEC or NHLF were seeded in precoated PureCol\u0026reg; bovine collagen 0.03 mg/mL (Advanced BioMatrix) 96 well plates the day before transfection with 50 ng FGF7 encoded mRNA (modFGF7), with full 5-methoxyuridine substitution (TriLink, CleanCap AG) complexed with Lipofectamine\u0026trade; 2000 (Invitrogen) at a mass ratio of 1:4. Supernatant containing secreted modFGF7 protein or media spiked with rhFGF7 (20 pg/\u0026micro;l), was transferred to hBECs every 24 hours for 72 hours before mechanical injury using a pipette tip. Wound closure was monitored over 72 hours using the JuLi\u0026trade; Stage Real-Time Cell History Recorder (NanoTek) and closure calculated using JuLi\u0026trade; STAT cell analysis software (v 2.0.0). Secreted modFGF7 protein in supernatant was quantified using a human FGF7 specific enzyme-linked immunosorbent assay kit (Sigma, RAB0188-1KT) according to the manufacturers protocol.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescent staining\u003c/h3\u003e\n\u003cp\u003eCells were fixed with 4% paraformaldehyde before permeabilization, blocking, and overnight incubation at 4\u0026deg;C with primary antibodies against Ki67 (Invitrogen, 14569882) or KTR5 (Invitrogen, PA1-37974) or FGF7R2-IIIb (R\u0026amp;D systems, MAB665-SP). After washing, cells were incubated with a species relevant goat IgG conjugated to either Alexa Fluor 488, 555 or 594 (Thermofisher, A-1100) and counterstained with DAPI (Invitrogen). Cells were imaged at 10x magnification using JuLi\u0026trade;.\u003c/p\u003e\n\u003ch3\u003eGene expression studies\u003c/h3\u003e\n\u003cp\u003eRNA was isolated using RNeasy Plus Micro Kit (Qiagen) and reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer\u0026rsquo;s protocols. Real-time quantitative PCR (RT-qPCR) was performed using Real Time PCR Systems, ViiA7 (Applied Biosystems, Software Version 1.2.3) or Bio-Rad CFX Opus 384 (CFX Maestro software, version 1.1). Reactions were set up by combining 0.3 \u0026micro;l TaqMan\u0026trade; gene expression assay (Applied Biosystems), 0.3 \u0026micro;l nuclease-free water, 3 \u0026micro;l TaqMan\u0026trade; Fast Advanced Master Mix (Applied Biosystems) with 2.4 \u0026micro;l cDNA in a 384-well format. Thermocycling conditions: 50\u0026deg;C/2 min, 95\u0026deg;C/20 sec, 40 cycles of 95\u0026deg;C/1 sec, 60\u0026deg;C/20 sec. Relative expression was calculated using the formula 2\u003csup\u003e\u0026minus;ΔCT\u003c/sup\u003e. TaqMan\u0026trade; gene expression assays used are as follows: \u003cem\u003eACTB\u003c/em\u003e: Hs01060665_g1, \u003cem\u003eAKT1\u003c/em\u003e: Hs00178289_m1, \u003cem\u003eCMYC\u003c/em\u003e: Hs00153408_m1, \u003cem\u003eFGFR2\u003c/em\u003e: Hs00256382_m1, \u003cem\u003eMAPK1\u003c/em\u003e: Hs01046830_m1, \u003cem\u003eMAPK3\u003c/em\u003e: Hs00385075_m1, \u003cem\u003eMTOR\u003c/em\u003e: Hs00234508_m1, \u003cem\u003ePIK3CA\u003c/em\u003e: Hs00180679_m1, \u003cem\u003ePLCG1\u003c/em\u003e: Hs06639798_s1.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed in GraphPad Prism 10 (Version 10.3.1 (464)). Statistical analyses are indicated in the figure legends and significance between groups was assessed with a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data from 2\u0026ndash;6 primary cell donors, with 2\u0026ndash;3 technical replicates each, are shown as mean and standard deviation unless otherwise indicated.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003erhFGF7 increases bronchial epithelial cell proliferation under physiological stress\u003c/h2\u003e\u003cp\u003eWhen seeded at low density, anchorage dependent epithelial cells can undergo anoikis due to loss of contact to neighbouring cells, preventing growth and proliferation. This phenomenon was observed in control KRT5\u0026thinsp;+\u0026thinsp;hBECs, where sub-optimal seeding density resulted in a lack of increased confluency up to 40 hours post-seeding (11.7%\u0026plusmn;2.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, hBECs treated with 5\u0026ndash;30 pg/\u0026micro;l of rhFGF7 demonstrated a dose-dependent increase in cell confluency, which was significant at all doses (5: P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 10: P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, 20: P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, 30: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), ranging from 19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8% at the 5 pg/\u0026micro;l dose, up to 25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0%, at 30 pg/\u0026micro;l. Although the 60 pg/\u0026micro;l dose significantly increased hBEC confluency compared to baseline (19.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), this was lower than that of the 30 pg/\u0026micro;l rhFGF7 dose suggesting lower doses are sufficient to stimulate hBEC proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The proportion of proliferating cells was quantified by immunofluorescent staining of Ki67 40 hours post-treatment. Cells treated with 5 pg/\u0026micro;l dose of rhFGF7 demonstrated a non-significant increase in Ki67 positive cells (54.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.7%) compared to PBS control (37.2\u0026thinsp;\u0026plusmn;\u0026thinsp;23.0%). Treatment with higher doses of 10\u0026ndash;60 pg/\u0026micro;l rhFGF7 facilitated a significant increase in the proportion of actively proliferating, Ki67 positive cells (57.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8% to 63.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6%; 10\u0026ndash;30 pg/\u0026micro;l: P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 60 pg/\u0026micro;l: P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003erhFGF7 improves survival of bronchial epithelial cells seeded at low density.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn agreement with the confluency assay, the physiological stressor of low seeding density promoted hBEC apoptosis as indicated by lactate dehydrogenase (LDH) release, with control cells secreting 115.7\u0026thinsp;\u0026plusmn;\u0026thinsp;11.7% LDH at 40 hours post-seeding relative to Triton X (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). When hBECs were treated with 10\u0026ndash;60 pg/\u0026micro;l rhFGF7, a significant reduction in cytotoxicity was observed (47.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3% \u0026minus;\u0026thinsp;40.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Cells treated with the lowest dose of 5 pg/\u0026micro;l rhFGF7 did not express reduced cytotoxicity, with LDH release of 103.3\u0026thinsp;\u0026plusmn;\u0026thinsp;56.9% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Together, these data suggest a range of 10\u0026ndash;30 pg/\u0026micro;l rhFGF7 may be protective against primary hBEC cell death \u003cem\u003ein vitro\u003c/em\u003e. A dose of 20 pg/\u0026micro;l was selected for all future experiments, we first confirmed that this dose would increase the proportion of live cells while reducing dead cells as expected based on previous results. The proportion of live cells following treatment with 20 pg/\u0026micro;l rhFGF7 was significantly higher (73.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) than that of control cells, where the majority were dead at the 40 hours post-seeding and live cell number was reduced to 19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). After confirming that FGF7 can impact hBEC proliferation and survival, next it was investigated whether synthetic mRNA encoding FGF7 could be translated in hBECs and impact wound closure compared to rhFGF7.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eProphylactic application of recombinant or mRNA derived FGF7 to hBEC monolayers improves repair.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEpithelial cells do not endogenously express FGF7, however when transfected with exogenous FGF7 mRNA, primary hBECs are capable of significant dose-dependent FGF7 translation and secretion (50 ng mRNA: 55.44\u0026thinsp;\u0026plusmn;\u0026thinsp;32.06 pg, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; 100 ng mRNA: 157.90\u0026thinsp;\u0026plusmn;\u0026thinsp;43.14 pg, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to non-transfected hBECs (0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 pg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Translation of FGF7 mRNA was transient with FGF7 protein levels peaking at 12\u0026ndash;24 hours post-transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To determine whether FGF7 influences hBEC wound repair, a prophylactic strategy was implemented as previously described\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Primary hBECs were treated with FGF7 secreted from mRNA transfected hBECs (modFGF7) or rhFGF7 and wounded 72 hours post-treatment. Wounded hBECs treated with modFGF7 (36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;21.1%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) or rhFGF7 (50.5\u0026thinsp;\u0026plusmn;\u0026thinsp;21.5%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) both demonstrated significantly increased wound closure at 36-hours post-injury compared to PBS treated wounds (15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.3%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). At 72-hours post-injury, treated cells achieved wound closure while control wounds remained unhealed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), possibly due to the additional contribution of increased survival and proliferation at the longer timepoint. The improvement in wound closure in hBECs that received FGF7 was accompanied by upregulation in genes that are key to re-epithelialisation during repair by mediation of genes associated with cell survival, growth, proliferation, and migration \u003cem\u003e(RAC1, CDH1, CTNNB1, PI3KCA, MAPK1/3, MTOR\u003c/em\u003e, and \u003cem\u003eCMYC).\u003c/em\u003e Interestingly, \u003cem\u003eFGFR2\u003c/em\u003e and \u003cem\u003ePLCG1\u003c/em\u003e were also upregulated in FGF7 treated and healed wounds suggesting activation of signal transduction through the FGF7 receptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Greater wound repair was observed in rhFGF7 treated wounds compared to modFGF7, and similarly higher expression of endogenous epithelial repair genes was observed with rhFGF7 treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIPF hBECs demonstrate active survival, proliferation, and migration that is not impacted by FGF7 treatment .\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter establishing positive responses to FGF7 in normal or \u0026lsquo;healthy\u0026rsquo; hBECs, we sought to investigate how hBECs from fibrotic lung would respond to the growth factor. First, it was confirmed that hBECs from IPF donors could translate \u003cem\u003eFGF7\u003c/em\u003e mRNA. Cells were transfected at two dose levels and resulted in significant and dose-dependent secretion of FGF protein as measured by ELISA over 72-hours (50ng: 31.44\u0026thinsp;\u0026plusmn;\u0026thinsp;8.36 pg, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 100 ng: 60.16\u0026thinsp;\u0026plusmn;\u0026thinsp;12.80 pg, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to non-transfected cells (0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 pg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, transient expression kinetics followed a similar pattern to healthy hBECS, with peak FGF7 detected between 12\u0026ndash;24 hours post-transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To determine if prophylactic FGF7 treatment could impact IPF hBEC migration in a wound closure assay, supernatant from \u003cem\u003eFGF7\u003c/em\u003e mRNA transfected cells or media spiked with rhFGF7 was applied to IPF hBECs prior to mechanical injury. Control, PBS treated IPF hBEC wounds were capable of notable wound closure 36 hours post-wounding (27.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and treatment with FGF7 did not improve closure (rhFGF7: 28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;19.5% and modFGF7: 16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;13.2%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Relative expression of key epithelial repair genes involved in proliferation, survival and migration were found to be largely unchanged in FGF7 treated wounds compared to PBS control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eIn a low seeding density assay, IPF-hBECs almost doubled in confluency in both rhFGF7 treated and PBS control conditions (20.9%8.1\u0026thinsp;\u0026plusmn;\u0026thinsp;and 18.8%\u0026plusmn;9.6, respectively) 40 hours post-seeding (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Cytotoxicity was measured by LDH release and confirmed that IPF hBECs were relatively resistant to cell death compared to healthy hBECs with only 27.4% (\u0026plusmn;\u0026thinsp;13.0) LDH release observed in PBS control and 23.9% (\u0026plusmn;\u0026thinsp;7.4) in rhFGF7 treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further explore the notable rates of proliferation and repair in IPF hBEC, and limited response to FGF7, baseline gene expression of hBECs from IPF and healthy donors cultured under standard conditions was compared. FGFR2 receptor gene expression was similar both in healthy and IPF hBECs confirmed by PCR and protein expression confirmed by immunostaining for FGFR2b against KRT5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, gene expression downstream of the FGF7 signalling axis were elevated in IPF hBECs compared to healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Expression of genes related to cell proliferation and pro-survival was significantly upregulated; \u003cem\u003ePLCG1\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), \u003cem\u003eMTOR\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), \u003cem\u003ePIK3CA\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and \u003cem\u003eAKT\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Expression of \u003cem\u003eCMYC, MAPK1\u003c/em\u003e, and \u003cem\u003eMAPK3\u003c/em\u003e was higher in IPF hBECs than in healthy controls, although not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Expression of \u003cem\u003eTGFB1\u003c/em\u003e, a potent suppressor of endogenous FGF7 production\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, was significantly higher in IPF hBECs (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDenudation and apoptosis of lung epithelium can occur during acute or chronic lung injury and dysfunctional repair is implicated in the pathogenesis of pulmonary diseases such as IPF\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and COPD\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. To investigate the impact of the epithelial mitogen FGF7 on hBECs \u003cem\u003ein vitro\u003c/em\u003e, low seeding density was employed as a physiological stressor to reduce cell-cell interaction, which inhibits cell growth and proliferation, ultimately leading to programmed cell death\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We hypothesised that rhFGF7 treatment of primary hBEC would enhance proliferation and survival under these conditions. In this study, rhFGF7 treatment enhanced healthy hBEC survival at low seeding densities. Cell proliferation was enhanced 2-fold compared to PBS control treated cells while cytotoxicity was reduced by 71%. Moreover, rhFGF7 treatment enhanced the proportion of live cells compared to PBS control treated cells. Balasooriya \u003cem\u003eet al\u003c/em\u003e report similar findings of FGF7\u0026rsquo;s ability to increase proliferation and growth of low-density murine airway basal cells compared to untreated cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In addition, Shyamsundar \u003cem\u003eet al\u003c/em\u003e utilised bronchoalveolar lavage (BAL) fluid from Palifermin treated subjects with LPS induced acute lung injury to treat alveolar epithelial cells \u003cem\u003ein vitro\u003c/em\u003e and demonstrated significant improvements in cell proliferation of the A549 adenocarcinoma cell line. However, the level of FGF7 in the BAL fluid of treated and untreated subjects was not quantified\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eLocalised and transient protein therapy is desirable when systemic toxicity is dose-limiting but where this is difficult to achieve with recombinant proteins, modified IVT mRNA may be an alternative modality for protein replacement. Primary hBECs do not endogenously express FGF7, therefore we established whether FGF7 encoded mRNA could be translated to functional protein by hBECs. For the first time, we demonstrate non-viral delivery of modified \u003cem\u003eFGF7\u003c/em\u003e mRNA generates dose dependent levels of FGF7 protein secreted from primary hBECs. However, variability in protein levels produced by different donors was observed which may impact therapeutic dosing of IVT mRNA. In addition, we transfected primary normal human lung fibroblasts (NHLF) with FGF7 mRNA and measured higher levels of secreted FGF7 post-transfection (Fig. S1) possibly due to NHLF being innate producers of this growth factor, and therefore more efficient at FGF7 translation and processing\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. As well as FGF7, fibroblasts secrete various mediators that augment the epithelial repair process such as interleukin 6 (IL-6)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, interleukin 1β (IL-1β)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and epidermal growth factor (EGF)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and dermal fibroblast conditioned media alone has been shown to enhance keratinocyte wound repair\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, therefore our study utilised supernatant of transfected hBEC for assessing the impact of modFGF7 on hBEC wound closure. This work represents the first demonstration that pre-treatment of primary hBECs with rhFGF7 or modFGF7 secreted from mRNA transfected hBECs, significantly improves wound closure following mechanical injury. Wang \u003cem\u003eet al\u003c/em\u003e recently reported FGF7 treatment improves wound closure and proliferation of mechanically injured 16HBE cells\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and Denzinger \u003cem\u003eet al\u003c/em\u003e reported similar findings with supernatants from FGF7-mRNA transfected keratinocytes improving wound closure of scratched keratinocyte monolayers\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe overall improvement in healthy hBEC wound closure was accompanied by upregulation in genes associated with epithelial repair and FGF7 signal transduction. Activation and dimerization of \u003cem\u003eFGFR2b\u003c/em\u003e is demonstrated by upregulation in \u003cem\u003ePLCG1\u003c/em\u003e, encoding adaptor protein phospholipase C-γ (PLCγ) which is recruited to the activated receptor to facilitate recruitment of downstream signalling molecules\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Increased expression of \u003cem\u003eRAC1\u003c/em\u003e, a key regulator of the cell cycle, cell-cell adhesion, and cell motility\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e as well as key genes involved in re-epithelisation and cell-cell adhesion, \u003cem\u003eCDH1\u003c/em\u003e, encoding E-cadherin, and \u003cem\u003eCTNNB1\u003c/em\u003e, encoding β-catenin, was observed in wounds with improved healing\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eActivation of the PI3K/AKT and MAPK pathways in FGF7 treated hBEC wounds was demonstrated by upregulation in \u003cem\u003ePIK3CA\u003c/em\u003e, \u003cem\u003eMAPK1\u003c/em\u003e, and \u003cem\u003eMAPK3.\u003c/em\u003e Interestingly, \u003cem\u003eAKT1\u003c/em\u003e and \u003cem\u003eMTOR\u003c/em\u003e upregulation was observed in rhFGF7 but not modFGF7 treated wounds potentially due to lack of quantification of modFGF7 protein level in the supernatants of mRNA transfected cells, preventing matched dosing. Upregulation of \u003cem\u003eMAPK1, MAPK3\u003c/em\u003e, and \u003cem\u003eCMYC\u003c/em\u003e expression was observed in both rhFGF7 and modFGF7 treated wounds at 72 hours post-injury thereby promoting cell survival and proliferation. Muyal \u003cem\u003eet al\u003c/em\u003e report similar findings of exogenous FGF7 mediated activation of signalling through the MAPK pathway in emphysematous mice with upregulated \u003cem\u003eMAPK1\u003c/em\u003e and \u003cem\u003eMAPK3\u003c/em\u003e expression. In addition, increased expression of \u003cem\u003eCMYC\u003c/em\u003e was also demonstrated in this study\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Interestingly, in response to FGF7 treatment, upregulation of \u003cem\u003eFGFR2\u003c/em\u003e was observed in healthy hBECs. Together, these data suggest improved primary hBEC wound repair in response to prophylactic FGF7 is mediated by activation of genes that promote cell survival, proliferation, and migration. Intracellular signal transduction is complex, with many genes involved in the regulation of cell fate and behaviour, thus we acknowledge that this study only interrogates a small panel of genes responsible for epithelial repair. Furthermore, we only assayed changes in gene expression at the endpoint of the injury assay therefore temporal changes in expression were not captured.\u003c/p\u003e\u003cp\u003eBronchial epithelial cells may contribute to fibrotic lung disease through various mechanisms including ectopic expansion of KRT5\u0026thinsp;+\u0026thinsp;BECs in distal lung\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and overexpression of transforming growth factor β1 (TGF-β1) which is a potent inducer of fibrosis and negative regulator of endogenous FGF7 production\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. A reduction in \u003cem\u003eFGFR2\u003c/em\u003e expression in regions undergoing remodelling in the IPF lung has also been observed\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We found no difference in baseline \u003cem\u003eFGFR2\u003c/em\u003e expression between healthy and IPF hBECs but did observe increased gene expression of \u003cem\u003eTGF-β1\u003c/em\u003e. When cells were seeded at low density, control IPF hBECs increased in confluency to a greater extent than control healthy hBECs and treatment with rhFGF7 did not impact proliferation of IPF hBECs. Moreover, wound closure of untreated IPF hBEC wounds was higher than that of healthy hBEC control wounds, and prophylactic treatment of IPF hBECs with FGF7 did not improve wound closure. Unlike healthy hBECs, expression of genes related to survival, proliferation and migration were unchanged between FGF7 treated and PBS control IPF hBECs, although a trend in upregulation of \u003cem\u003eFGFR2\u003c/em\u003e and \u003cem\u003eTP63\u003c/em\u003e was observed in some donors (Fig. S2). Due to limitations in IPF cell numbers, increasing doses of FGF7 could not be tested on IPF-HBECs which may require higher doses to elicit a response. Finally, we acknowledge that submerged, monolayer culture of IPF hBECs is not representative of the spatial and temporal heterogeneity of IPF where areas of early and established fibrosis are present\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Nevertheless, it has previously been reported that hBECs\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e and fibroblasts from IPF lungs\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e have increased baseline pAKT/PI3K signalling leading to increased cell proliferation and survival, consequently inhibition of this pathway in fibroblasts is a pharmacological target to reduce fibrosis\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. However, IPF hBECs also undergo apoptosis after inhibition of PI3K/mTOR\u003csup\u003e47\u003c/sup\u003e, and off-target systemic epithelial cytotoxicity due to non-specific inhibition remains unknown. Consequently, there may be rationale for further exploration of FGF7 as a protective growth factor during pharmacological interventions that induce epithelial apoptosis such as non-selective PI3K/mTOR inhibition or bleomycin chemotherapy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis study demonstrates the feasibility of exogenous FGF7 encoded mRNA as a novel therapeutic strategy for repair of normal primary bronchial epithelial cells. Moreover, we demonstrate that exogenous FGF7 can induce \u003cem\u003eFGFR2\u003c/em\u003e gene expression and activate intracellular signalling that promotes primary hBEC proliferation, migration, and survival. Application to other diseases where impaired epithelial repair is caused by dysregulated FGFR2b signal transduction should be investigated.\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors would like to acknowledge funding from the UKRI Advanced Therapies Network (ref: CiC013), Medical Research Council (MR/W028433/1) and the Rosetrees Trust (ref: PhD2022\\100016). \u0026nbsp;The work was supported by the National Institute for Health \u0026amp; Care Research through the Imperial Biomedical Research Centre.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors have no relevant financial or non-financial competing interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAsha Patel, Philip Molyneaux and Skye Quinn contributed to the study conception and design. Material preparation, data collection and analysis were performed by Skye Quinn, Asha Patel, Alexander Jenkins and Rafaela Konstantinidi. The first draft of the manuscript was written by Skye Quinn and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe datasets generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eWritten informed consent was obtained from all subjects, and the study was approved by the local Research Ethics Committee (15/SC/0101 and 20/SC/0142).\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMiki, T. \u003cem\u003eet al.\u003c/em\u003e Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e89\u003c/strong\u003e, (1992).\u003c/li\u003e\n\u003cli\u003eZhang, X. \u003cem\u003eet al.\u003c/em\u003e Receptor Specificity of the Fibroblast Growth Factor Family. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e281\u003c/strong\u003e, (2006).\u003c/li\u003e\n\u003cli\u003eLiberti, D. 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T. \u003cem\u003eet al.\u003c/em\u003e A randomised, placebo-controlled study of omipalisib (PI3K/mTOR) in idiopathic pulmonary fibrosis. \u003cem\u003eEuropean Respiratory Journal\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, (2019).\u003c/li\u003e\n\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":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fibroblast growth factor-7, lung repair, modified mRNA, Human bronchial epithelial cells, idiopathic pulmonary fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-6939652/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6939652/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFibroblast growth factor 7 (FGF7) is a potent and specific epithelial mitogen that can modulate alveolar repair however, the impact on human bronchial epithelial cells (hBECs) is limited. This study characterised repair responses of hBECs from healthy and fibrotic lungs to exogenous FGF7 supplementation.\u003c/p\u003e\n\u003cp\u003eWhen healthy hBECs were cultured under a physiological stressor of low seeding density, treatment with recombinant human FGF7 (rhFGF7) reduced cytotoxicity, and increased survival and proliferation. Cell migration was assessed using a scratch assay, where pre-treatment of hBECs with rhFGF7 significantly increased wound closure (50.5%, p\u0026lt;0.001) compared to control (15.3%), accompanied by upregulation of FGF signalling genes including mTOR, PIK3CA and MAPK3. To explore modified mRNA as an alternative protein supplementation strategy for wound repair, it was found that hBECs were able to secrete dose responsive levels of FGF7 following mRNA transfection (modFGF7), and when applied to a monolayer of hBECs, wound closure was significantly improved compared to control (36.1%, p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eIn contrast, when hBECs from idiopathic pulmonary fibrosis (IPF) donors were cultured at low density or injured by scratch, untreated cells were capable of notable survival and wound closure (27.1%) that was not improved by rhFGF7 or modFGF7 treatment. It was found that baseline expression of genes associated with proliferation and survival including PIK3CA, AKT1 and MTOR was higher in IPF hBEC. This study demonstrates a role of FGF7 in proliferation, survival and migration of healthy hBECs but requires careful assessment dependant on disease context.\u003c/p\u003e","manuscriptTitle":"Fibroblast growth factor-7 promotes repair of primary human bronchial epithelial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 15:46:44","doi":"10.21203/rs.3.rs-6939652/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2025-07-08T10:08:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-24T14:04:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Medicine","date":"2025-06-23T11:20:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b8ff371e-8994-4bec-b7c8-0c52a11ecd44","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-10T15:46:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 15:46:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6939652","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6939652","identity":"rs-6939652","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}