Intrinsic Regeneration Mechanisms of Murine Lacrimal Gland Epithelial Cells for Therapeutic Applications in Aqueous- Deficient Dry Eye Disease

Intrinsic Regeneration Mechanisms of Murine Lacrimal Gland Epithelial Cells for Therapeutic Applications in Aqueous- Deficient Dry Eye Disease | 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 Intrinsic Regeneration Mechanisms of Murine Lacrimal Gland Epithelial Cells for Therapeutic Applications in Aqueous- Deficient Dry Eye Disease Ingrid Zahn, Hannah Franke, Sophie Gleixner, Nicolas Blavet, Jana Dietrich, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8077136/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Aqueous-deficient dry eye disease (ADDE) results from lacrimal gland dysfunction leading to tear film instability and chronic ocular surface inflammation. Despite the gland’s known regenerative potential, the intrinsic molecular mechanisms enabling epithelial recovery remain poorly understood. This study aimed to characterize intrinsic regeneration processes of murine lacrimal gland epithelial cells (mLGECs) and identify pathways with therapeutic potential. Methods Primary mLGECs were isolated from neonatal C57BL/6J mice and characterized by RT-PCR, immunocytochemistry, and β-hexosaminidase secretion assays. Acute injury was induced by brief exposure to 20% ethanol. Cell viability, apoptosis, and transcriptional responses were analyzed during early recovery phases. Pathway enrichment was conducted using Gene Ontology and KEGG annotations. Regeneration-associated growth factors were validated by quantitative PCR, ELISA, and functional assays assessing cell viability and wound closure in the presence or absence of the MAPK inhibitor SCH772984. Results Cultured mLGECs preserved epithelial and lacrimal-specific markers and maintained regulated secretory activity. RNA-seq two hours post-injury revealed robust activation of MAPK signaling, accompanied by strong upregulation of regenerative ligands including Hbegf, Areg, Ngf, and Gdnf. Transient protein induction of HBEGF and AREG was confirmed. Functionally, HBEGF, AREG, NGF, and GDNF markedly enhanced epithelial survival, ATP recovery, and wound closure, while MAPK inhibition abolished regeneration, demonstrating pathway dependence. Conclusion These results identify MAPK-dependent mechanisms as central to the intrinsic regenerative response of lacrimal gland epithelial cells following acute injury. HBEGF and AREG emerged as key mediators driving epithelial survival and proliferation. The findings provide a mechanistic basis for developing defined growth factor–based therapies as standardized, safe, and effective alternatives to blood-derived treatments for patients with severe ADDE. aqueous-deficient dry eye disease dry eye disease epithelial repair growth factors lacrimal gland regeneration MAPK signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Dry Eye Disease (DED) is a multifactorial condition affecting the ocular surface and tear film. Clinically, patients often experience persistent foreign body sensation in the eyes, burning, itching, recurrent conjunctivitis, and visual impairments, which markedly impairs daily life. DED can be classified into two subtypes: evaporative dry eye (EDE) and aqueous-deficient dry eye (ADDE). The evaporative form is primarily associated with meibomian gland dysfunction, whereas the hypovolemic form arises from lacrimal gland dysfunction [ 1 ].The most common cause of Sjogren-associated ADDE is immune-mediated inflammation, characterized by infiltration of the lacrimal gland by immune cells, predominantly CD4 + T-cells, leading to progressive destruction of the lacrimal gland [ 2 , 3 ]. The lacrimal gland is predominantly composed of acinar epithelial cells, which are organized into functional units known as acini. Additional cell types include ductal epithelial cells forming the excretory duct system and myoepithelial cells providing contractile support to the acini. The interstitial space also contains immune cells such as IgA-secreting plasma cells, macrophages, as well as T and B lymphocytes [ 4 ]. Acinar epithelial cells produce a protein-rich aqueous secretion, that is modified by ductal epithelial cells during transport to the ocular surface. This secretion constitutes the main component of the mucin-aqueous phase of the tear film and contains inorganic salts, along with more than 1,500 proteins and peptides. The most abundant proteins include lactoferrin, lysozyme, tear lipocalin, and IgA [ 1 ]. Like other secretory glands, the lacrimal gland possesses lifelong regenerative capacity [ 5 ]. Animal models have shown that lacrimal glands are capable of complete regeneration in vivo following acute and severe injury [ 6 – 8 ]. However, the molecular and cellular mechanisms underlying this process remain poorly understood. A recent study on salivary gland regeneration revealed that acinar epithelial cells possess self-regeneration potential [ 9 ]. These cells actively proliferate to replace those that have been lost due to physiological turnover or induced damage. In the present study, we investigate the intrinsic regeneration mechanisms of lacrimal gland acinar epithelial cells. Using transcriptome analysis of mouse acinar epithelial cells during active regeneration, we identified key molecular pathways involved in this process. 2. Materials and methods 2.1 Mice Male and female C57BL/6J mice were obtained from Charles River Laboratories (Wilmington, MA, USA) and maintained under a 12:12-hour light: dark cycle with food and water provided ad libitum. Extraorbital lacrimal glands were excised from 1- to 3-day-old mice sacrificed by decapitation. All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in compliance with FELASA recommendations for animal experimentation. The procedures were approved and registered by the Animal Welfare Office of Friedrich Alexander University (TS-12/14 Anatomie II, 26 August 2020). 2.2 Murine Lacrimal Gland Epithelial Cells (mLGECs) The excised lacrimal glands were washed with cold phosphate-buffered saline (PBS) (Sigma-Aldrich, Taufkirchen, Germany) before being minced with a scalpel. The minced lacrimal glands were transferred to a collagen-coated (125-50; Merck, Darmstadt, Germany) culture dish and allowed to attach to the surface. Subsequently, culture medium (DMEM-F12, #D8062, Merck, Darmstadt, Germany), supplemented with 10% fetal calf serum (FCS, S0615, Bio&SELL, Feucht, Germany), 1% penicillin/streptomycin/amphotericin B (#A5955, Merck), 0.1% hydrocortisone (#H0888, Merck), 0.1% cholera toxin (#C8052; Merck), 1% sodium bicarbonate solution (#25080060, Thermo Fisher Scientific, Waltman, USA), 1% adenine (#A2786, Merck), 1% T3/transferrin solution (#T8158, Merck), 0.1% insulin solution (#I5500, Merck), and 0.0002% mouse epidermal growth factor (EGF, PMG8044, Gibco, Waltham, MA, USA), was added. For routine cultivation, cells that migrated out of the minced lacrimal gland tissue were enzymatically detached using trypsin-EDTA (0.05%; #T3924; Merck) and subcultured in fresh collagen-coated dishes. To remove contaminating fibroblasts, a selective detachment step was performed using 0.01% trypsin-EDTA. The culture medium was replaced every two days. 2.3 RNA Purification for Sequencing mLGECs were dissociated using lysis buffer containing 10% SDS, 10% glycerol, 0.005% bromophenol blue, 62.5 mM Tris-HCl (pH 6.8), and 5% β-mercaptoethanol (added fresh) and stored at −80 °C. RNA was extracted using the RNeasy Mini Kit (#74104, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). Extracted RNA samples were stored at −80 °C prior to sequencing (n = 6). 2.4 RNA Sequencing and Data Analysis High-throughput RNA-Seq data were prepared using Lexogen Quantseq FWD kit for Illumina with polyA selection and sequenced on Illumina NovaSeq sequencer (run length 1x75 nt). Bcl files were converted to Fastq format using bcl2fastq v. 2.20.0.422 Illumina software for basecalling. Quality check of raw single-end fastq reads was carried out by FastQC [10]. The adapters and quality trimming of raw fastq reads was performed using Trimmomatic v0.39 [11] with settings CROP:250 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:5 MINLEN:35. Trimmed RNA-Seq reads were mapped against the mouse genome (mm38) and Ensembl GRCm38 v.93 annotation using STAR v2.7.3a [12] as splice-aware short read aligner and default parameters except --outFilterMismatchNoverLmax 0.66 and --twopassMode Basic. Quality control after alignment concerning the number and percentage of uniquely- and multi-mapped reads, rRNA contamination, mapped regions, read coverage distribution, strand specificity, gene biotypes and PCR duplication was performed using several tools namely RSeQC v4.0.0 [13], Picard toolkit v2.25.6 [14], Qualimap v.2.2.2 [15]. The differential gene expression analysis was calculated based on the gene counts produced using featureCounts from Subread package v2.0 [16] and further analyzed by Bioconductor package DESeq2 v1.34.0 [17]. Data generated by DESeq2 with independent filtering were selected for the differential gene expression analysis due to its conservative features and to avoid potential false positive results. Genes were considered as differentially expressed based on a cut-off of adjusted p-value ≤ 0.05 and log2(fold-change) ≥1 or ≤-1. Clustered heatmaps were generated from selected top differentially regulated genes using R package pheatmap v1.0.12 [18], volcano plots were produced using ggplot2 v3.3.5 package [19] and MA plots were genereted using ggpubr v0.4.0 package [20]. 2.5 RNA extraction, cDNA synthesis and reverse-transcription PCR Total RNA was extracted from mouse lacrimal glands or mLGECs and subsequently transcribed into cDNA as previously described ADDIN CitaviPlaceholder{{"$id":"1","$type":"SwissAcademic.Citavi.Citations.WordPlaceholder, SwissAcademic.Citavi","Entries":[{"$id":"2","$type":"SwissAcademic.Citavi.Citations.WordPlaceholderEntry, SwissAcademic.Citavi","Id":"8c413e96-19e8-4c2b-9f8a-0dfa97c76880","RangeLength":3,"ReferenceId":"8529341a-8b3c-4f4b-9d50-330a681e8395","PageRange":{"$id":"3","$type":"SwissAcademic.PageRange, SwissAcademic","EndPage":{"$id":"4","$type":"SwissAcademic.PageNumber, 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To verify cDNA integrity, β-actin PCR was performed on all samples. Each reaction was carried out using Taq DNA Polymerase (Thermo Fisher Scientific), 100 mM dNTPs (Thermo Fisher Scientific), and 2 × 10 pmol gene-specific primers (Table 1), following the manufacturer’s instructions. The PCR products were then separated on a 2% agarose gel, stained with GelRed Nucleic Acid Gel Stain (Linaris, Dossenheim, Germany), and analyzed via electrophoresis alongside a Low Range DNA Ladder (#SM0383, Thermo Fisher Scientific). 2.6 Quantitative real-time PCR (qPCR) Quantitative real-time PCR analysis was performed using a LightCycler® 480 (Roche, Basel, Switzerland) in combination with Takyon™ MasterMix (Eurogentec, Seraing, Belgium), following the manufacturer’s instructions. All reactions were conducted in triplicates, with a no-template control included. Primer efficiency was calculated for each primer used (Table 1), and data analysis was carried out according to Pfaffl ADDIN 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} [23] . 2.7 Immunofluorescence After two washes with PBS, cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes. Permeabilization was then performed using 0.1% Triton X, followed by additional PBS washes. The cells were subsequently incubated with 10% normal serum for one hour, then incubated overnight with the diluted primary antibody (Table 2). The following day, the cells were washed again with PBS and incubated for one hour with the secondary antibody (Table 2) in PBS. This was followed by a 5-minute incubation with DAPI (Sigma-Aldrich) and a final mounting using Fluorescence Mounting Medium (Agilent Technologies, Santa Clara, USA). 2.8 β-Hexosaminidase Assay To assess the secretory activity of mLGECs, the cells were stimulated with either 0.1 mM carbachol or 1 mM dibutyryl-cAMP (dbcAMP) in serum-free medium for 1 hour. Following stimulation, the supernatants were collected, the cells were lysed, and both the supernatants (SN) and lysates (L) were stored at –80 °C until further analysis. β-hexosaminidase activity was measured using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (M2133, Sigma-Aldrich) as a substrate, and fluorescence was quantified at 450/365 nm (emission/excitation). The amount of β-hexosaminidase released was calculated as substrate turnover in the supernatant relative to the lysate, using the following formula: beta-hexosaminidase release = (ΔE SN * Factor SN )/(ΔE L * Factor L ) [24]. 2.9 Ethanol (EtOH) Induced Damage of mLGECs mLGECs were left untreated or stimulated with 12.5 ng/mL recombinant mouse glial cell line-derived neurotrophic factor (GDNF; Thermo Fisher, #4504450UG), amphiregulin (AREG; Bio-Techne, #989-AR-100/CF), nerve growth factor (NGF; Bio-Techne, #1156-NG-100/CF), or heparin-binding epidermal growth factor-like growth factor (HBEGF; BIOZOL, #MCE-HY-P7194-50UG) for 2 hours, or treated with 10 nM SCH772984, an extracellular signal-regulated kinase (ERK) inhibitor (MedChemExpress, Monmouth Junction, USA) for 24 hours. Cells were then washed with PBS and exposed to 20% EtOH (v/v) for 1 minute. EtOH was aspirated, cells were washed with PBS, and fresh medium was added. Intracellular ATP levels were measured at 0, 1, 2, 4, 6, and 24 hour post-treatment. Samples for Annexin V/PI staining and RNA-seq were collected at 0, 2, and 6 hours. 2.10 Intracellular ATP Assay After stimulation mLGECs were washed with PBS and incubated with a 1:1 mixture of CellTiter-Glo® 3D Reagent (#G9683, Promega, Walldorf, Germany) and PBS for 10 minutes at room temperature. Luminescence was then measured using a CLARIOstar Plus plate reader (BMG Labtech, Ortenberg, Germany) according to the manufacturer's protocol. 2.11 Annexin-V/PI for Flow Cytometry Quantification of life, dead, and apoptotic cells was performed using FITC-conjugated annexin V and propidium iodide (PI) staining, followed by flow cytometric analysis. Treated and untreated mLGECs (2 × 10 6 cells/mL) were collected, washed with PBS, and resuspended in 500 μL binding buffer. The cells were then stained with 10 μg/mL FITC-conjugated annexin V (#A13199, Thermo Fisher Scientific) and 50 μg/mL PI for 30 minutes at 4 °C in the dark. Samples were analyzed using a CytoFLEX S Flow Cytometer (Beckman Coulter, Krefeld, Germany), and data were processed using FlowJo software (FlowJo LLC; version 10.1). 2.12 Wound Healing Assay mLGECs were seeded at a density of 2 × 10 5 cells/mL into a µ-Dish 35 mm containing a 2-Well Culture-Insert (#81175, ibidi, Gräfelfing, Germany). After 24 hours of attachment, cells were stimulated with different growth factors (12.5 ng/mL; listed in Section 2.9, Ethanol (EtOH)-Induced Damage of mLGECs) for 2 hours, or with SCH772984 for 24 hours. Subsequently, the insert was removed, and the gap area was imaged at 0, 24, 48, and 72 hours using an inverted microscope (CKX53; Evident, Bad Kreuznach, Germany). Images were analyzed with cellSense Standard software version 4.2.1 (Evident). 2.13 Enzyme-linked Immunosorbent Assay For protein extraction, untreated and EtOH treated cells were detached using trypsin, washed with PBS, and lysed in Triton buffer (1% Triton X-100 in PBS) supplemented with protease and phosphatase inhibitors (2 µl/ml) for 30 minutes on ice. Total protein concentrations were determined using a BCA Protein Assay (#A55864, Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative analysis of Hb-EGF and amphiregulin concentrations was performed using commercial ELISA kits (Mouse Amphiregulin DuoSet ELISA, #DY989; Mouse HB-EGF DuoSet ELISA, #DY8239-05; R&D Systems, Minneapolis, USA) following the protocols provided by the manufacturer. 2.14 Statistics The data are presented as mean values with standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 10 (version 10.1.2, GraphPad Software Inc., San Diego, USA). Statistical significance was defined as a p-value of ≤ 0.05. 3. Results 3.1 Transcriptomic profiling of mLGECs A comparative analysis of the transcriptomic profiles of primary mLGECs (for isolation see Supplementary Figure 1 ) and native murine lacrimal gland tissue was conducted, which revealed a distinct difference in gene expression ( Figure 1A ). Among the top 20 differentially expressed genes, those associated with structural and contractile functions were strongly suppressed in mLGECs. Additionally, stress- and immune-related transcripts were prominently activated, together with genes involved in inflammatory, stress, and hormonal responses, as well as in metabolic processes ( Supplemental Table 1 ). Gene Ontology (GO) domain overrepresentation analysis supported these findings ( Figure 1B ). Among the upregulated categories, the most significantly enriched biological processes included the acute-phase response (Fold Enrichment 27.6, p = 4.7×10⁻¹²) and inflammatory response (6.0, p = 5.4×10⁻¹²). Enriched cellular components were dominated by the extracellular region (4.4, p = 2.0×10⁻³¹) and extracellular space (3.9, p = 1.2×10⁻²⁶), whereas enriched molecular functions included cytokine activity (9.0, p = 5.0×10⁻¹⁴) and growth factor activity (10.8, p = 4.3×10⁻¹²). In contrast, downregulated categories were dominated by contractile and muscle-associated terms. Biological processes included muscle contraction (33.1, p = 3.7×10⁻²⁰) and sarcomere organization (28.9, p = 3.2×10⁻¹²). Cellular components such as the Z-disc (14.1, p = 1.1×10⁻¹⁴) and I-band (38.4, p = 5.1×10⁻¹¹) were significantly reduced. Molecular functions associated with contractility, including actin filament binding (6.4, p = 2.9×10⁻⁷) and actin binding (29.7, p = 1.4×10⁻⁶), were likewise suppressed. Using a transcriptome atlas of the mouse lacrimal gland, a more detailed examination of stromal and epithelial subpopulations was conducted by averaging log2 fold-changes across their marker genes (unweighted mean per subgroup) ( Figure 1C ). All cell type populations were reduced in cultured mLGECs compared with native tissue, including fibroblasts (−1.43), endothelial (−6.49), mural (−1.07), lymphoid (−1.15), and myeloid cells (−2.73). Similarly, epithelial clusters such as acinar (−1.54), ductal (−2.30), and myoepithelial cells (−1.70) showed global downregulation. These results indicate a uniform loss of stromal and epithelial programs, consistent with dedifferentiation during adaptation to in vitro culture. 3.2 Expression of epithelial and gland-specific markers Epithelial and lacrimal gland specific markers were assessed across passages P1 to P3 by RT-PCR, using native lacrimal gland tissue (mLG) and 3T3 fibroblasts as controls, and additionally by immunocytochemistry at P1 ( Figure 2A, B ). Bands were detected for Aqp5 (144 bp), Rab3d (130 bp), Lyz1 (100 bp), Ltf (191 bp), Krt5 (177 bp), Foxc1 (171 bp), Sox10 (147 bp), Pax6 (245 bp), Acta2 (211 bp), and Vim (327 bp) across passages P1–P3 and native lacrimal gland tissue, while Ltf and Pax6 were absent in 3T3 cells (Figure 2A). Semi-quantitative analysis normalized to Actb indicated a slight decrease of epithelial markers from P1 to P3. Protein-level validation by immunocytochemistry showed membrane-associated AQP5 staining, cytoplasmic granular staining for LTF, cytoplasmic KRT5 staining, nuclear FOXC1/2, and α-SMA was detected in the cytoplasm. VIM staining was absent in mLGECs but strongly expressed in 3T3 fibroblasts ( Figure 2B ). Functional analysis of secretory activity demonstrated that β-hexosaminidase release increased upon stimulation. Both carbachol (mean difference −0.1417, p = 0.0066) and dibutyryl cAMP (db-cAMP) (mean difference −0.1847, p = 0.0004) induced significantly higher secretion compared to controls ( Figure 2C ), confirming at least a partial preservation of regulated exocytosis and secretory capacity in cultured mLGECs. 3.3 Acute injury and early regeneration Acute injury was induced by exposing mLGECs to 20% ethanol for 1 minute, followed by incubation in either serum-free or serum-containing medium ( Figure 3A ). Cell viability was assessed immediately prior to ethanol exposure and at 0, 1, 2, 4, and 24 hours post-treatment. A significant decline of viability to 40.95 ± 16.56% to the baseline value (p < 0.0001) was observed immediately after exposure, followed by partial recovery at 1 hour (55.04 ± 6.09%) and further increase at 2 hours (72.49 ± 11.54%) under serum conditions. At 48 hours (data not shown), viability exceeded baseline levels (24 h: 171.30 ± 39.96%; 48 h: 287.80 ± 116.28%), with minimal differences between treated and untreated cells, indicating full recovery. As serum withdrawal reduced growth in untreated controls, subsequent experiments were performed in serum-containing medium ( Figure 3B ). Apoptosis was assessed by Annexin V and propidium iodide staining to validate regulated cell death as the driving force behind reduced cell viability. Immediately after ethanol exposure, the proportion of living cells decreased significantly (p < 0.0001) with a concomitant increase in dead cells (p < 0.0001). At this point no apoptotic cells were detected and probably other forms of cell death occur. At 2 hours, apoptotic cells were significantly elevated (p < 0.0001) while the fraction of living cells further declined (p = 0.0065). By 6 hours, the distribution of live, apoptotic, and dead cells returned to baseline levels, indicating a consistent recovery trend. This kinetics mirrored the ATP-based viability measurements, showing an early decline followed by gradual recovery ( Figure 3C ). To gain an unbiased insight into pathways that could induce cell recovery and be protective for mLGECs, we performed total mRNA sequencing at the 2-hour time point in serum-containing medium. This timepoint was selected, as it represents the early recovery phase, which is characterized by partial restoration of viability and the peak in apoptotic signaling. 3.4 Transcriptomic analysis of intrinsic regeneration mechanisms after acute injury Total mRNA sequencing revealed that among the top 20 differentially expressed genes, stress- and autophagy-related genes were strongly upregulated together with immune and interferon-associated transcripts. In contrast, a developmental regulator was downregulated ( Figure 4A, Supplemental Table 2 ). GO overrepresentation analysis of upregulated genes highlighted significantly enriched biological processes, including leukocyte cell–cell adhesion (Fold Enrichment 14.2, p = 1.1 × 10⁻⁶) and response to virus (6.1, p = 1.3 × 10⁻⁵). The top enriched cellular components were extracellular region (Fold Enrichment 1.8, p = 9.8 × 10⁻⁶) and cell projection (1.9, p = 1.9 × 10⁻⁴). At the molecular function level, significant terms comprised sequence-specific double-stranded DNA binding (Fold Enrichment 2.6, p = 2.4 × 10⁻⁵) and receptor agonist activity (6.3, p = 8.5 × 10⁻⁵). KEGG pathway analysis revealed enrichment of MAPK signaling (2.3, p = 4.3×10⁻⁴), TNF signaling (3.1, p = 1.5 × 10⁻³) and IL-17 signaling (3.2, p = 3.6 × 10⁻³) ( Figure 4B, top ). Downregulated categories were dominated by transcription- and development-related processes. The most significant biological processes were regulation of transcription from RNA polymerase II promoter (Fold Enrichment 4.5, p = 9.1 × 10⁻⁴) and positive regulation of endothelial cell migration (43.8, p = 1.9 × 10⁻³). At the molecular function level, significantly reduced terms included RNA polymerase II transcription factor activity, sequence-specific DNA binding (Fold Enrichment 5.7, p = 2.3 × 10⁻⁴) and growth factor activity (16.2, p = 1.3 × 10⁻²). ( Figure 4B, bottom ). Based on this analysis, TNF and MAPK signaling were selected for detailed follow-up analyses. 3.5 MAPK and TNF Signaling Pathways: Regeneration Mechanisms After Acute Injury Acute injury activated both TNFR1 and TNFR2-mediated arms of TNF signaling, with increased expression across adaptor and kinase nodes and their immediate-early outputs. Downstream effectors of inflammation and survival were induced, including the Fos/Jun family ( Fos log2FC 2.87, p = 3.9×10⁻⁵; Jun 2.26, p = 1.5×10⁻⁸), NF-κB regulators ( Rela 1.45, p = 3.7×10⁻⁵²; Nfkbia 1.10, p = 3.9×10⁻⁹), inflammatory cytokines ( Tnf 2.22, p = 0.013), chemokines involved in leukocyte recruitment ( Ccl5 4.94, p = 4.3×10⁻⁷⁸; Cxcl10 4.75, p = 4.7×10⁻⁵⁵; Cxcl2 2.78, p = 1.4×10⁻⁶), and adhesion/vascular genes ( Icam1 1.38, p = 1.1×10⁻⁷; Sele 2.06, p = 0.048; Edn2 2.14, p = 0.0044). Additional immune regulators were upregulated, including Sqstm1 (1.68, p = 5.1×10⁻⁶⁹), Traf1 (1.62, p = 0.045), and Il18r1 (4.33, p = 1.3×10⁻⁶). In addition to Tnf , multiple growth factor-receptor-associated ligands of the MAPK pathway were differentially expressed. Significant upregulation was observed for Hbegf (2.69, p = 9.2×10⁻⁶⁶), Areg (2.95, p = 1.1×10⁻²²), Ngf (2.88, p = 4.9×10⁻⁴³), Gdnf (3.16, p = 5.2×10⁻²¹), Ereg (1.06, p = 2.6×10⁻⁹), Fgf9 (2.18, p = 0.0046), Efna1 (1.31, p = 2.9×10⁻¹¹), Angpt2 (1.20, p = 0.045), Pdgfa (1.25, p = 2.8×10⁻⁴³), Kitl (1.14, p = 3.3×10⁻²⁵), Epha2 (1.61, p = 1.3×10⁻⁵), Kdr (1.48, p = 3.3×10⁻⁴), and Met (1.53, p = 2.0×10⁻¹⁴). In contrast, Fgf2 (–2.19, p = 9.9×10⁻²⁸), Fgf10 (–1.07, p = 9.0×10⁻⁹), and Egf (–2.31, p = 1.4×10⁻⁶) were significantly downregulated. Furthermore, the cascade elements and downstream transcriptional targets were also affected. Significant upregulation was observed for Kit (2.50, p = 8.0×10⁻⁴), Mknk2 (1.31, p = 1.2×10⁻²⁹), Atf3 (5.54, p = 3.8×10⁻³⁹), Creb5 (2.01, p = 8.3×10⁻²⁸), Rasgrf1 (2.36, p = 0.0084), and Cacna2d2 (2.75, p = 0.0019) (Figure 5A, B ). To validate differential MAPK-pathway regulation after acute injury, the expression of selected components was quantified by qPCR. Genes encoding signaling ligands ( Hbegf, Areg, Fgf9, Gdnf, Ngf, Efna1, Egf ), cascade elements ( Kit, Epha2 ), and downstream targets ( Creb5, Jun, Fos ) were examined. Two hours after injury, all genes except Fgf9, Efna1, and Kit were significantly altered and Efna1 and Kit showed a tendency toward upregulation (Figure 5C). Comparing mean normalized expression (MNE) in untreated samples revealed relatively high basal levels of Epha2 (1.6×10⁻¹ ± 3.3×10⁻²), Hbegf (6.3×10⁻² ± 2.5×10⁻²), and Ngf (2.9×10⁻² ± 7.0×10⁻³), whereas Areg (1.4×10⁻⁵ ± 6.3×10⁻⁶) and Kit (5.4×10⁻⁶ ± 1.4×10⁻⁶) exhibited low baseline expression ( Supplementary Figure 2A ). For all confirmed significant signaling ligands, expression was further profiled over a 24-hour period following acute ethanol injury. Hbegf was strongly induced at 2 hours (p = 0.0008) and peaked at 4 hours (p < 0.0001). Areg was significantly upregulated at 2 hours (p = 0.0007) and 4 hours (p = 0.0016). Gdnf showed significant elevation at 4 hours (p < 0.0001), and Ngf peaked at 4 hours (p < 0.0001). In contrast, Egf was significantly downregulated at 2 hours (p < 0.0001), with a weaker but still detectable reduction at 4 hours (p = 0.037) ( Supplementary Figure 2B ). 3.6 Growth Factor–Mediated Regeneration of mLGECs After Acute Injury To evaluate whether MAPK growth factors contribute to regeneration after ethanol-induced injury, we performed cell viability and wound-healing assays. Dose–response analyses confirmed high-affinity activity for HBEGF, NGF, GDNF, and AREG (Kd < 1 ng/ml, saturation at 12.5 ng/ml) and identified a non-cytotoxic concentration of 10 nM for the MAPK inhibitor SCH772984 (IC₅₀ 32.7–98.5 nM across donors) ( Figure 5A, Supplementary Figure 2C ). Following ethanol exposure, all MAPK growth factors enhanced viability recovery, whereas SCH772984 caused a progressive decline ( Figure 6A, Supplementary Figure 3 ). Viability decreased by more than 50% at 0 hours under all conditions, excluding acute protective effects. AREG showed the earliest improvement at 2 hours (p = 0.0217). By 4 hours, all factors significantly promoted recovery (p < 0.01) and by 24 hours, HBEGF exerted the strongest effect (+66%, p < 0.0001), followed by AREG (+44%), GDNF (+22%), and NGF (+15%). Pairwise comparisons confirmed differential efficacy, with HBEGF outperforming GDNF (p = 0.0004) and NGF (p < 0.0001), and AREG showing greater activity than NGF (p = 0.0140). Wound-healing assays further substantiated these findings. All MAPK growth factors accelerated wound closure relative to untreated controls (p < 0.0001), whereas SCH772984 markedly impaired closure (−38%, p < 0.0001) (Figure 5B). Among growth factors, HBEGF was significantly more effective than GDNF (+30%, p = 0.0009) and NGF (+27%, p = 0.0035) ( Figure 6B) . Finally, protein-level analyses aligned with transcriptional findings. Both HBEGF and AREG were transiently upregulated within the first two hours after ethanol exposure before returning to baseline, paralleling their mRNA profiles. HBEGF showed a robust peak at 2 hours (p = 0.0003), while AREG exhibited a weaker but significant induction at the same time point (p = 0.027). Across all time points, HBEGF levels remained consistently higher than those of AREG, consistent with gene-expression profiles ( Figure 6C ). 4. Discussion 4.1 Characterization of Acinar Epithelial Cells from Murine Lacrimal Glands (mLGEC) Transcriptomic profiling of mLGECs revealed a pronounced downregulation of contractile programs. This could reflect skeletal muscle contamination during dissection due to the gland’s anatomical proximity to the masseter muscle [ 25 ] or selective vulnerability of myoepithelial cells during enzymatic isolation and culture [ 26 ]. Nevertheless, the persistence of Acta2/α-SMA expression suggests that myoepithelial cells are not completely absent, but rather that contractile pathways are broadly suppressed under in vitro conditions. In contrast to the loss of contractile signatures, stress- and immune-related genes were strongly upregulated in cultured mLGECs. Among the genes, Sgk1 , encoding for a well-characterized stress-responsive kinase, has been shown to be rapidly induced under diverse cell-stress conditions [ 27 ]. Similarly, increased expression of Ptx3 , Cxcl5 , Spp1 , and Serpine1 suggests enhanced immunomodulatory activity [ 28 ]. The induction of Cyp1b1 is consistent with metabolic adaptation and survival mechanisms, as CYP1B1 has been shown to promote proliferation, inhibit apoptosis, and facilitate cell-cycle progression during in vitro differentiation [ 29 ]. Collectively, these findings indicate that mLGECs engage a broad stress- and immune-related program as part of their adaptation process, likely in response to the absence of physiological signaling cues and exposure to artificial medium components [ 30 , 31 ]. Using the mouse lacrimal gland transcriptome atlas [ 32 ], we examined marker genes for stromal populations and epithelial cell clusters. Stromal cells included fibroblasts that form the interstitial connective tissue; vascular cells comprising endothelial and mural cell populations across venous, arterial, and capillary vessels [ 33 ]; and resident immune cells of lymphoid and myeloid lineages that mediate immune surveillance and defense within the interstitium [ 4 ]. Epithelial clusters comprised of acinar cells secreting aqueous tear components, ductal epithelial cells forming the excretory duct system, and myoepithelial cells providing contractile support to acini. Notably, both stromal and epithelial subpopulations exhibited global downregulation in culture, a pattern consistent with partial dedifferentiation, a well‑recognized phenomenon following cell isolation and adaptation to two‑dimensional culture systems [ 34 , 35 ]. Despite the global transcriptomic shift observed in culture, mLGECs retained key epithelial and lacrimal gland specific features. Core functional markers, including Aqp5 and Rab3d , essential for water transport and vesicle trafficking [ 36 , 37 ], as well as secretory proteins Ltf and Lyz1 [ 38 ], remained expressed, with AQP5 and LTF confirmed at the protein level. The basal epithelial identity was further supported by Krt5 expression, while developmental regulators Foxc1 , Sox10 , and Pax6 [ 39 – 41 ] were preserved, albeit with reduced Pax6 levels compared to native tissue. Notably, the absence of VIM protein in mLGECs, despite transcript detection, contrasted with its strong expression in 3T3 fibroblasts arguing against fibroblast contamination. Functionally, mLGECs retained regulated exocytosis, as demonstrated by β-hexosaminidase release, indicating preserved secretory function despite partial dedifferentiation and stress adaptation. Collectively, these findings indicate that while culture conditions induce transcriptional remodeling, mLGECs retain key hallmarks of lacrimal gland epithelial identity and maintain their functionality during early passages. 4.2. Acute Injury Responses and Intrinsic Regeneration Mechanisms Our findings demonstrate that ethanol exposure induces a rapid injury response in mLGECs, followed by early signs of recovery. Ethanol is widely used as a nonspecific cytotoxic agent, as it partitions into lipid bilayers, increases membrane fluidity and permeability, disrupts barrier properties, and denatures proteins, collectively causing ATP depletion and apoptotic or lytic cell death in epithelial cells [ 42 – 44 ]. The sharp decline in viability and increase in apoptosis within the first hours after exposure mirror injury kinetics reported in corneal epithelial cells [ 44 ], confirming ethanol as a robust model of acute-injury. Importantly, recovery dynamics within 24–48 hours, even under serum-free conditions, suggest that mLGECs have an inherent intrinsic regenerative capacity as seen in vivo [ 8 , 45 ]. This resilience likely reflects activation of conserved stress-response and repair programs that support epithelial survival after acute cytotoxic insults [ 46 ]. Transcriptomic profiling reinforced this interpretation, revealing strong induction of stress-, immune-, and repair-associated pathways alongside suppression of transcriptional and developmental programs. Upregulation of canonical stress regulators such as Sqstm1 , Fnip1 , and Ndrg1 together with GO biological-process enrichment for response to virus, inflammatory response, and leukocyte cell–cell adhesion suggests the induction of autophagy, metabolic adaptation, and energy-sensing pathways. This is consistent with reports that epithelial cells engage conserved stress responses to maintain survival after injury [ 47 , 48 ]. Concurrent induction of chemokines like Ccl5 and interferon-stimulated genes such as Ifit1 , along with enrichment for the GO cellular components cytoplasm, cell projection, and axoneme, reflects early immunomodulatory responses, aligning with studies showing rapid cytokine release and immune activation in ocular epithelia following acute insults [ 49 , 50 ]. Importantly, the expression of repair-associated factors, including Klf5 and Hbegf , highlights that regenerative mechanisms are engaged already during the early recovery phase, consistent with evidence that growth-factor signaling via EGFR ligands promotes epithelial wound healing [ 51 , 52 ]. This pattern mirrors findings from animal models of lacrimal gland injury, which report immediate inflammatory and immune responses, including early upregulation of Il1b (IL-1β) [ 45 , 53 ]. The downregulation of Id3 and associated transcriptional regulators suggests a transient suppression of differentiation and developmental processes [ 54 ], likely reflecting a trade-off between energy-demanding transcriptional activity and immediate stress adaptation [ 55 , 56 ], aligning with GO enrichment analyses showing reduced transcription-related processes. Together, these observations support a model in which acute ethanol injury elicits a tightly coordinated early response program in mLGECs. Rapid stress adaptation and innate immune activation, which occur alongside the prompt engagement of intrinsic repair pathways and the transient recession of differentiation. 4.3 Growth Factor–Driven Regeneration in the Injured Lacrimal Gland Acute epithelial injury to mlGECs rapidly activates both TNF and MAPK signaling, underscoring the interplay of inflammatory and regenerative mechanisms in the early repair phase. Upregulation of TNFR1- and TNFR2-associated transcripts, along with downstream effectors such as Fos , Jun , NF-κB regulators, cytokines ( Il1b , Il6 , Tnf ), chemokines ( Ccl2 , Ccl5 , Cxcl2 ) and adhesion molecules ( Icam1 , Sele , Vcam1 , Vegfa , Edn ), reflects a “damage-control” response that promotes rapid immune-cell recruitment, vascular stabilization, and barrier protection while loosening the matrix to facilitate repair [ 57 ]. Although TNF signaling clearly contributes to regeneration, its strong proinflammatory and cytotoxic properties limits its suitability as a therapeutic target [ 58 ]. In contrast, the MAPK pathway emerged as a key driver of epithelial regeneration. Multiple growth-associated ligands were differentially regulated, including upregulation of Hbegf , Areg , Ngf , Gdnf , Fgf9 , and Ereg , while Fgf2 , Fgf10 , and Egf were downregulated. EGF-like factors such as HBEGF, AREG, EREG, and EGF typically promote cell proliferation and tissue renewal and can selectively modulate lacrimal gland functions [ 51 ]. Notably, Egf expression declined following ethanol injury, despite exogenous EGF in the culture medium supporting cell survival and, at higher concentrations, enhancing regeneration. This reduction likely reflects negative feedback regulation of endogenous Egf in the presence of extracellular ligand, a well-characterized mechanism in EGFR signaling [ 59 , 60 ]. Thus, while mLGECs suppress their own Egf production, EGFR remains responsive, and regeneration appears to be preferentially driven by inducible ligands such as HBEGF and AREG [ 61 , 62 ]. In addition to the EGF family, FGF family members, including FGF2 , FGF9 , and FGF10 , regulate cell growth, differentiation, and development. In the lacrimal gland, mesenchymal FGF10 signaling via epithelial FGFR2b drives gland induction and branching during development [ 40 , 63 ] and, supports epithelial proliferation in adult or regenerative contexts [ 64 ]. The observed downregulation of Fgf10 following EtOH injury likely reflects stage-specific dynamics, with early injury and inflammatory responses preceding later regenerative proliferation, and does not contradict its established role [ 53 ]. Neurotrophic factors, including NGF and GDNF promote cell survival and regeneration, particularly in neuronal and epithelial tissues. In the lacrimal gland, NGF likely functions in an autocrine/paracrine manner to maintain epithelial function and secretion [ 65 ]. In parallel, the GDNF-family ligand neurturin is critical for parasympathetic innervation of the lacrimal gland: neurturin-deficient (NRTN⁻/⁻) mice display markedly reduced parasympathetic nerve fibers, diminished tear production and a DED phenotype [ 66 ]. Taken together, these findings position MAPK signaling as a central regulator of lacrimal gland homeostasis, stress adaptation, and regeneration. Consistent with this, modulation of the p38 and JNK pathways in models of radiation-induced lacrimal gland damage markedly reduced tissue damage and improved glandular function, underscoring the importance of MAPK signaling in tissue repair [ 67 , 68 ]. Our data extend these findings by demonstrating that MAPK signaling is not only intrinsically activated in lacrimal gland cells following acute injury but can also be further enhanced by stimulation with HBEGF, AREG, NGF, GDNF and EGF. Although no acute protective effects were observed, enhanced regenerative recovery was evident, with HBEGF and AREG exerting the strongest effects. Importantly, pharmacological MAPK inhibition abrogated recovery, highlighting the essential role of this pathway in epithelial repair. Clinically, these results align with regenerative therapies already employed in patients with severe DED. Autologous serum (AS) eye drops, enriched in growth factors such as EGF, TGF-β, NGF, IGF, and PDGF, are widely used and improve tear stability, corneal epithelial healing, and patient symptoms, particularly in Sjogren’s syndrome and ocular surface disorders [ 69 , 70 ]. Similarly, platelet-rich plasma (PRP) offers higher concentrations of platelets and growth factors than AS and can be administered via direct injection into the human lacrimal gland. Recent clinical studies demonstrated that intraglandular PRP injections significantly enhanced tear secretion, reduced ocular surface damage, and alleviated symptoms in severe DED, highlighting their regenerative potential at the glandular level [ 71 – 73 ]. However, both AS and PRP are limited by donor variability, labor-intensive preparation, risks of contamination or instability, and complex regulatory classification as blood-derived biologics, restricting broader clinical application [ 69 , 74 ]. These limitations have sparked growing interest in replacing blood-derived products with standardized cocktails of defined growth factors, aiming for greater consistent efficacy and safety. Comparative studies emphasize that the regenerative activity of PRP largely depends on its content of factors like EGF, NGF, IGF, and PDGF, underscoring the need to identify the key components and their optimal concentrations [ 70 , 75 ]. Moreover, recent perspectives explicitly call for the development of controlled and optimized PRP-derived formulations, highlighting the need for standardization or substitution with defined growth factor combinations [ 75 ]. In this context, our study provides critical insight by revealing growth factors intrinsically upregulated following acute injury in isolated mLGECs, offering a rational framework for designing targeted therapeutic cocktails to enhance lacrimal gland regeneration. Although pan-MAPK activation is undesirable due to its association with tumorigenesis and inflammation [ 76 , 77 ], a transient and pathway-specific stimulation could potentially promote lacrimal gland self-renewal. 5. Conclusion Our study demonstrates that murine lacrimal gland acinar epithelial cells (mLGECs) undergo transcriptional remodeling in culture, characterized by downregulation of contractile programs and partial dedifferentiation, while retaining key lacrimal gland and epithelial markers and preserving their secretory capacity. Acute ethanol injury triggered a rapid stress and inflammatory response, including induction of Fos , Jun , NF-κB regulators, cytokines, and chemokines, reflecting a damage-control phase that stabilizes the tissue and prepares it for repair. Simultaneously, regenerative pathways were activated, with strong upregulation of MAPK ligands such as Hbegf , Areg , Ngf , and Gdnf . Functional assays identified HBEGF and AREG as the most potent mediators of recovery, while MAPK inhibition completely abolished regeneration, underscoring the essential role of this pathway. Clinically, these findings align with AS and PRP therapies in severe DED, whose efficacy depends on growth factor content but is limited by its variability, preparation complexity, and regulatory challenges. By defining the intrinsically induced growth factors during lacrimal gland epithelial acinar cell regeneration after acute injury, our study establishes a rational preclinical basis on the road to developing standardized, safe, and effective growth factor-based therapies, opening a viable alternative to blood-derived products and new opportunities for targeted regenerative strategies in human lacrimal gland injury and disease in the future. Abbreviations ADDE, Aqueous-Deficient Dry Eye; AQP5, Aquaporin 5; AREG, Amphiregulin; ATP, Adenosine Triphosphate; cAMP, Cyclic Adenosine Monophosphate; db-cAMP, Dibutyryl Cyclic Adenosine Monophosphate; DED, Dry Eye Disease; DMEM, Dulbecco’s Modified Eagle Medium; EGF, Epidermal Growth Factor; ELISA, Enzyme-Linked Immunosorbent Assay; EtOH, Ethanol; FCS, Fetal Calf Serum; FGF, Fibroblast Growth Factor; FOXC1/2, Forkhead Box Protein C1/C2; GDNF, Glial Cell Line-Derived Neurotrophic Factor; GO, Gene Ontology; HB-EGF (HBEGF), Heparin-Binding EGF-Like Growth Factor; IL, Interleukin; KEGG, Kyoto Encyclopedia of Genes and Genomes; KRT5, Keratin 5; LG, Lacrimal Gland; LTF, Lactotransferrin; LYZ1, Lysozyme 1; MAPK, Mitogen-Activated Protein Kinase; mLG, Murine Lacrimal Gland; mLGECs, Murine Lacrimal Gland Epithelial Cells; mRNA, Messenger Ribonucleic Acid; NF-κB, Nuclear Factor Kappa B; NGF, Nerve Growth Factor; PAX6, Paired Box 6; PBS, Phosphate-Buffered Saline; PCR, Polymerase Chain Reaction; PI, Propidium Iodide; PRP, Platelet-Rich Plasma; qPCR, Quantitative Polymerase Chain Reaction; RNA-seq, RNA Sequencing; RT-PCR, Reverse Transcription Polymerase Chain Reaction; SCH772984, Selective MAPK/ERK Kinase Inhibitor; SOX10, SRY-Box Transcription Factor 10; TNF, Tumor Necrosis Factor; TNFR, Tumor Necrosis Factor Receptor; VIM, Vimentin Declarations Financial Disclosure The authors have no proprietary or commercial interest in any materials discussed in this manuscript. FP receives royalties from Elsevier for the 25 th Ed. of the anatomy atlas “Sobotta” and FP for the 3 rd of the “Sobotta Textbook of Anatomy”. The present work was performed in fulfillment of the requirements of obtaining the degree Dr. rer. nat. (IZ). Funding This work was funded by: Ernst und Berta Grimmke Foundation (project 6/21) to JD; in part by the German Research Foundation (DFG, grants PA738/15-1 and PA738/15-2) to FP; by a Sicca-Research-Award (Sicca-Förderpreis) of the Dry Eye and Ocular Surface Disease Resort of the Professional Association of German Ophthalmologists to IZ. Conflicts of Interests All authors declare no conflict of interests. Acknowledgment The authors gratefully acknowledge the valuable support provided by Maike Hemmerlein, Anke Fischer-Gößwein (molecular biology), Hong Nguyen (immunohistochemistry) and Jörg Pekarsky (graphics). Core Facility Genomics and Core Facility Bioinformatics of CEITEC Masaryk University are gratefully acknowledged for their support with obtaining scientific data presented in this paper. Author contributions Conceptualization: I.Z., J.D. and P.A.; methodology: I.Z.; validation: I.Z.; formal analysis: I.Z., N.B. and P.A.; investigation: I.Z. and H.F.; resources: P.A. and F.P.; data curation: I.Z. and H.F.; writing—original draft: I.Z.; writing—review and editing: all authors; visualization: I.Z. and N.B.; supervision: F.P. and P.A.; project administration: F.P. and P.A.; funding acquisition: J.D. and F.P. All authors have read and agreed to the published version of the manuscript. Availability of data and materials The RNAseq data are accessible under the ArrayExpress accession E-MTAB-16008. References Bron AJ, Paiva CS de, Chauhan SK, Bonini S, Gabison EE, Jain S, et al. Tfos dews II pathophysiology report. The ocular surface. 2017;15:438–510. 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Primer (q)PCR Forward Reverse Amplicon 18S qPCR GGT GCA TGG CCG TTC TTA TGC CAG AGT CTC GTT CGT TA 69 bp Acta2 PCR CAT CAC CAA CTG GGA CGA CA AGA GGC ATA GAG GGA CAG CA 211 bp Actb PCR GAT CCT CAC CGA GCG CGG CTA CA GCG GAT GTC CAC GTC ACA CTT CA 298 bp Aqp5 PCR CAT GAA CCC AGC CCG ATC TT CTC AGC GAG GAG GGG AAA AG 144 bp Areg qPCR AGA TAC ATC GAG AAC CTG GAG G AGA GAC AAA GAT AGT GAC AGC TAC 150 bp Creb5 qPCR CGA TAA CAG CCA TGC AGA AAG CGA TGA GGA TGT AGT GAT GGT G 128 bp Cstb PCR GAT CTG TCT TCA GCT TCT CCG TCG ATT CAA GCT GGG ACT TC 134 bp Efna1 qPCR CAT CTC CAA ACC TAT CTA CCA TCA G TGC AAA ACC TGT ACT TCC GG 147 bp Egf qPCR AGA AAC ACC AAG ACC CCA AG TGT GCC CAT TCC ATC TAT GTG 149 bp Epha2 qPCR TGC CAG CGT CAG TAT TAA CC TTC TTG CGG TAG GTG ACT TC 141 bp Fgf9 qPCR AAC TGG GCG GTT GGA ACA TA TGT CAC AGA AGT GGG CTG AA 119 bp Fos qPCR TAC TAC CAT TCC CCA GCC GA GCT GTC ACC GTG GGG ATA AA 113 bp Foxc1 PCR GGG CTT TCG TCT ATG ACT GTA G TTC TGT TCG CTG GTG TGA G 171 bp Gdnf qPCR TGA AGA CCA CTC CCT CGG GCT TGT TTA TCT GGT GAC CTT TTC 150 bp Hbegf qPCR CCC CTA TAC ACA TAT GAC CAC AC CAA CTT CAC TTT CTC TTC ACT TTC C 150 bp Jun qPCR TGG GCA CAT CAC CAC TAC AC TCT GGC TAT GCA GTT CAG CC 119 bp Kit qPCR TGT GGC TAA AGA TGA ACC CTC ACA CTC CAG AAT CGT CAA CTC 135 bp Krt5 PCR AGA GGC TGA GTC CTG GTA C ATG GCG TTC TGG AGG TTG 177 bp Ltf PCR ATC TCT GTG CCC TGT GTA TTG GTT CCT AGC CCA CTC TTC AG 191 bp Lyz1 PCR GGA CTA GTG AGC TGT GCC TG TTG CTC CTG TGG TTA TTG GCT 100 bp Ngf qPCR CCC AAT AAA GGT TTT GCC AAG G TTG CTA TCT GTG TAC GGT TCT G 137 bp Pax6 PCR CAG ATG CAA AAG TCC AGG TGC TTG GCT GCT AGT CTT TCC CG 245 bp Pdgfra PCR GTT GCC TTA CGA CTC CAG ATG TCA CAG CCA CCT TCA TTA CAG 143 bp Rab3d PCR CTA TGC CGA TGA CTC CTT CAC ATG AGC AGG AAA CCC ATA GC 180 bp Sox10 PCR AGC AGG CTG GAC ACT AAA CC AAA GGG GCA GCG ATG TGT TA 147 bp Vim PCR TGA GAT CGC CAC CTA CAG GA TTG CGC TCC TGA AAA ACT GC 327 bp 18S : 18S ribosomal RNA; Acta2 : actin alpha 2 or alpha smooth muscle actin; Actb : β-actin; Aqp5: aquaporin 5; Areg : amphiregulin; Creb5 : cyclic AMP-responsive element-binding protein 5; Cstb : cystatin B; Efna1 : ephrin A1; Egf : epidermal growth factor; Epha2 : EPH receptor A2; Fgf9 : fibroblast growth factor 9; Fos : Fos proto-oncogene; Foxc1 : Forkhead Box C1; Gdnf : Glial cell line-derived neurotrophic factor; Hbegf: heparin-binding EGF-like growth factor; Jun : Jun proto-oncogene; Kit : Kit proto-oncogene; Krt5 : keratin 5; Ltf : lactoferrin; Lyz1 : lysozyme 1; Ngf : nerve growth factor: Pax6 : paired box 6; Pdgfra : platelet derived growth factor receptor alpha; Rab3d : RAB3D, member RAS oncogene family; Sox10 : SRY (sex determining region Y)-box 10; Vim : vimentin Table 2. Antibodies. Antibody Conjugation Solution # Manufacturer Goat Anti-Mouse IgG Alexa 488 1:500 A11029 Thermo Fisher Scientific, Waltman, USA Donkey Anti-Rabbit IgG Alexa 488 1:1000 A21206 Thermo Fisher Scientific, Waltman, USA α-SMA - 1:1000 ab124964 Abcam, Cambridge, UK AQP5 - 1:25 sc-514022 Santa Cruz Biotechnology, Dallas, USA CYT5 - 1:50 sc-32721 Santa Cruz Biotechnology, Dallas, USA LTF - 150 PA5-95513 Thermo Fisher Scientific, Waltman, USA FOXC1/2 - 1:100 CPA3482 Cohesion Biosciences, London, UK VIM 1:200 1NP3.1 Carl Roth, Karlsruhe, Germany α-SMA: or alpha smooth muscle actin; AQP5: aquaporin 5; CYT5: cytokeratin 5; LTF: lactoferrin; FOXC1/2: forkhead box protein C1/C2; VIM: vimentin Additional Declarations No competing interests reported. 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14:07:32","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":247940,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8077136/v1/668b45e60d6a8e43fd087b32.html"},{"id":97261771,"identity":"4f843101-4935-44bc-aaa6-73d45bd17bdb","added_by":"auto","created_at":"2025-12-02 14:07:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":281123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA-seq comparison of native murine lacrimal gland tissue (mLG) and isolated lacrimal gland cells (mLGECs).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eHeatmap of the top 20 significantly differentially expressed genes with hierarchical clustering of genes and samples (n=6 different donors; primary mLGECs, left; native mLG, right). Colors indicate normalized expression (z-scores). \u003cstrong\u003e(B) \u003c/strong\u003eGene Ontology over-representation analysis performed with DAVID. Up- and down-regulated gene sets were analyzed separately; Bars display the p-value of the top five enriched terms are shown for each GO domain: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). \u003cstrong\u003e(C)\u003c/strong\u003e Log₂ fold change of established lacrimal-gland cell-type marker genes for stromal populations (fibroblast, endothelial, mural, lymphoid, myeloid; left) and epithelial clusters (acinar, ductal, myoepithelial; right). Positive values indicate higher expression in isolated cells (mLGECs) relative to native tissue (mLG). Colored vertical lines indicate the unweighted mean log₂ fold change across marker genes within each stromal (left) and epithelial (right) group.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8077136/v1/0fcc624058f87cbde4dc03ad.png"},{"id":97367340,"identity":"b4c835d5-1128-4e65-bb82-79d8a3a8d246","added_by":"auto","created_at":"2025-12-03 16:18:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":456082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLacrimal gland specific markers and secretory function of cultured mLGECs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMarkers:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Aqp5\u003c/em\u003e: aquaporin-5 (AQP5), \u003cem\u003eRab3d\u003c/em\u003e: Ras-related protein Rab-3D (RAB3D), \u003cem\u003eLtf\u003c/em\u003e: lactotransferrin (LTF), \u003cem\u003eLyz1\u003c/em\u003e: lysozyme-1 (LYZ1), \u003cem\u003eKrt5\u003c/em\u003e: keratin-5 (KRT5), \u003cem\u003eFoxc1\u003c/em\u003e: forkhead box C1 (FOXC1), \u003cem\u003eSox10\u003c/em\u003e: SRY-box transcription factor 10 (SOX10), \u003cem\u003ePax6\u003c/em\u003e: paired box 6 (PAX6), \u003cem\u003eActa2\u003c/em\u003e: alpha-smooth-muscle actin (α-SMA), \u003cem\u003eVim\u003c/em\u003e: vimentin (VIM), and \u003cem\u003eActb\u003c/em\u003e: beta-actin (ACTB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e RT-PCR of marker genes across passages P1–P3 of the mLGECs, native lacrimal gland (mLG), and 3T3 fibroblasts (a cell line derived from mouse embryonic fibroblasts used as feeder layer). The lower panel shows semiquantitative analysis normalized to Actb with (n = 3 biological replicates). Data are mean ± SEM. \u003cstrong\u003e(B)\u003c/strong\u003e Immunocytochemistry for AQP5, LTF, KRT5, FOXC1/2, α-SMA, and VIM. Positive signal appears as green fluorescence, nuclei are counterstained with DAPI (blue). 3T3 cells serve as positive control for VIM. \u003cstrong\u003e(C)\u003c/strong\u003e β-Hexosaminidase release assay. Stimulation with carbachol and dibutyryl cAMP (db-cAMP) increased secretion relative to control, indicating preserved regulated exocytosis. n = 10 biological replicates; two-way ANOVA with Dunnett’s multiple comparisons test; **p ≤ 0.01, ***p ≤ 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8077136/v1/90196d174f87d823643176e6.png"},{"id":97367976,"identity":"ed139f37-5a65-42e8-b829-454b84d976b4","added_by":"auto","created_at":"2025-12-03 16:21:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcute ethanol (EtOH) injury and early recovery of mLGECs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Experimental workflow. mLGECs at ~80% confluence were exposed to 20% ethanol for 1 minute and then returned to proliferation medium. Readouts were obtained at 0 2, 4, 6, and 24 hours after injury.\u003cstrong\u003e (B)\u003c/strong\u003eRelative viability/intracellular ATP of mLGECs with and without 20% EtOH treatment, following serum free (−FCS) or serum containing (+FCS) media incubation over 24 hours, measured by CellTiter-Glo 2.0 assay. The dotted line marks baseline (untreated = 100%). Data are mean ± SEM; each point represents the mean of technical triplicates for one donor.\u003cstrong\u003e (C)\u003c/strong\u003e Annexin V/propidium iodide analysis of live, apoptotic, and dead cells at 0, 2, and 6 hours after EtOH treatment (stacked bar plot, top) with representative dot plots (bottom) of untreated, 0 hour, and 2 hour samples; n = 6 biological replicates; two-way ANOVA with Dunnett’s multiple comparisons test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8077136/v1/2beb854635bdfff3ed5fb159.png"},{"id":97261779,"identity":"d1cb872a-a333-4855-a9c6-fa72b619ece1","added_by":"auto","created_at":"2025-12-02 14:07:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":149159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic response of mLGECs two hours after ethanol (EtOH) treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Heatmap of the top 20 significantly differentially expressed genes between 20 % EtOH-treated (+EtOH) and untreated (−EtOH) mLGECs, with hierarchical clustering of genes and samples. Colors indicate normalized expression (z-scores). \u003cstrong\u003e(B)\u003c/strong\u003e Over-representation analysis of the resulting gene sets. Up- and down-regulated lists were analyzed separately. Bars display the p-value of the top five enriched terms are shown for each GO domain: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) as well as enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8077136/v1/35a58e8c5d2fa69109f315c7.png"},{"id":97261774,"identity":"fbc0fdaa-dc8b-4a4a-80a9-14674cef69d3","added_by":"auto","created_at":"2025-12-02 14:07:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":452449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential expression of genes in the KEGG TNF and classical MAPK signaling pathways after ethanol treatment of mLGECs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Visualization of differentially expressed genes (log₂ fold change ≥ 1/2 or ≤ –1/2) within the TNF and classical MAPK signaling pathways according to KEGG annotation. Significantly regulated genes are indicated with red (upregulated) or blue (downregulated) stars. \u003cstrong\u003e(B)\u003c/strong\u003elog₂ fold change and adjusted p-values of genes highlighted in (A). Bars represent mean log₂ fold change values, with significance thresholds indicated. \u003cstrong\u003e(C)\u003c/strong\u003e Validation of selected MAPK-pathway genes by quantitative PCR after 2 hours\u003c/p\u003e\n\u003cp\u003eexposure of mLGECs to 20% ethanol. Data are mean ± SEM; n = 6 biological replicates; paired t-test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8077136/v1/c1be892ca268d705f782afba.png"},{"id":97368208,"identity":"093d0efe-554b-41ab-8125-09446a310a0a","added_by":"auto","created_at":"2025-12-03 16:21:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":591447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth factor–induced regeneration and protein expression following acute ethanol injury. (A)\u003c/strong\u003e Relative viability/ intracellular ATP of mLGECs with or without 20% ethanol injury in EGF-free medium, supplemented with growth factors (12.5 ng/ml EGF, HBEGF, NGF, GDNF, AREG) or the MAPK kinase inhibitor SCH772984 (10 nM) over 24 hours. n = 9 biological replicates \u003cstrong\u003e(B)\u003c/strong\u003eWound-healing assay after 24 and 48 hours under the same conditions. n = 6 biological replicates. (C) HBEGF and AREG protein levels in mLGECs after treatment with 20% ethanol (EtOH), shown over a 24 hours time course n = 4-6 biological replicates; Data are mean ± SEM; two-way ANOVA with Dunnett’s multiple comparisons test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8077136/v1/7f03b5c041889bc0ebfacee2.png"},{"id":98804962,"identity":"d88781b5-a22c-4fb1-a909-c6020ebc1b20","added_by":"auto","created_at":"2025-12-22 14:23:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3661647,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8077136/v1/950ed32f-4f59-47a5-8d17-31c1a7cbe17f.pdf"},{"id":97261772,"identity":"a2c7b46b-62fb-4a93-90fc-6a4697953c5d","added_by":"auto","created_at":"2025-12-02 14:07:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":673839,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8077136/v1/a936b550d6539686b4677e68.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intrinsic Regeneration Mechanisms of Murine Lacrimal Gland Epithelial Cells for Therapeutic Applications in Aqueous- Deficient Dry Eye Disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDry Eye Disease (DED) is a multifactorial condition affecting the ocular surface and tear film. Clinically, patients often experience persistent foreign body sensation in the eyes, burning, itching, recurrent conjunctivitis, and visual impairments, which markedly impairs daily life. DED can be classified into two subtypes: evaporative dry eye (EDE) and aqueous-deficient dry eye (ADDE). The evaporative form is primarily associated with meibomian gland dysfunction, whereas the hypovolemic form arises from lacrimal gland dysfunction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].The most common cause of Sjogren-associated ADDE is immune-mediated inflammation, characterized by infiltration of the lacrimal gland by immune cells, predominantly CD4\u003csup\u003e+\u003c/sup\u003e T-cells, leading to progressive destruction of the lacrimal gland [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe lacrimal gland is predominantly composed of acinar epithelial cells, which are organized into functional units known as acini. Additional cell types include ductal epithelial cells forming the excretory duct system and myoepithelial cells providing contractile support to the acini. The interstitial space also contains immune cells such as IgA-secreting plasma cells, macrophages, as well as T and B lymphocytes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Acinar epithelial cells produce a protein-rich aqueous secretion, that is modified by ductal epithelial cells during transport to the ocular surface. This secretion constitutes the main component of the mucin-aqueous phase of the tear film and contains inorganic salts, along with more than 1,500 proteins and peptides. The most abundant proteins include lactoferrin, lysozyme, tear lipocalin, and IgA [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Like other secretory glands, the lacrimal gland possesses lifelong regenerative capacity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Animal models have shown that lacrimal glands are capable of complete regeneration \u003cem\u003ein vivo\u003c/em\u003e following acute and severe injury [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the molecular and cellular mechanisms underlying this process remain poorly understood. A recent study on salivary gland regeneration revealed that acinar epithelial cells possess self-regeneration potential [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These cells actively proliferate to replace those that have been lost due to physiological turnover or induced damage. In the present study, we investigate the intrinsic regeneration mechanisms of lacrimal gland acinar epithelial cells. Using transcriptome analysis of mouse acinar epithelial cells during active regeneration, we identified key molecular pathways involved in this process.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1\u003c/strong\u003e \u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale and female C57BL/6J mice were obtained from Charles River Laboratories (Wilmington, MA, USA) and maintained under a 12:12-hour light: dark cycle with food and water provided ad libitum. Extraorbital lacrimal glands were excised from 1- to 3-day-old mice sacrificed by decapitation. All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in compliance with FELASA recommendations for animal experimentation. The procedures were approved and registered by the Animal Welfare Office of Friedrich Alexander University (TS-12/14 Anatomie II, 26 August 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Murine Lacrimal Gland Epithelial Cells (mLGECs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe excised lacrimal glands were washed with cold phosphate-buffered saline (PBS) (Sigma-Aldrich, Taufkirchen, Germany) before being minced with a scalpel. The minced lacrimal glands were transferred to a collagen-coated (125-50; Merck, Darmstadt, Germany) culture dish and allowed to attach to the surface. Subsequently, culture medium (DMEM-F12, #D8062, Merck, Darmstadt, Germany), supplemented with 10% fetal calf serum (FCS, S0615, Bio\u0026amp;SELL, Feucht, Germany), 1% penicillin/streptomycin/amphotericin B (#A5955, Merck), 0.1% hydrocortisone (#H0888, Merck), 0.1% cholera toxin (#C8052; Merck), 1% sodium bicarbonate solution (#25080060, Thermo Fisher Scientific, Waltman, USA), 1% adenine (#A2786, Merck), 1% T3/transferrin solution (#T8158, Merck), 0.1% insulin solution (#I5500, Merck), and 0.0002% mouse epidermal growth factor (EGF, PMG8044, Gibco, Waltham, MA, USA), was added. For routine cultivation, cells that migrated out of the minced lacrimal gland tissue were enzymatically detached using trypsin-EDTA (0.05%; #T3924; Merck) and subcultured in fresh collagen-coated dishes. To remove contaminating fibroblasts, a selective detachment step was performed using 0.01% trypsin-EDTA. The culture medium was replaced every two days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e\u003cem\u003eRNA Purification for Sequencing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emLGECs were dissociated using lysis buffer containing 10% SDS, 10% glycerol, 0.005% bromophenol blue, 62.5 mM Tris-HCl (pH 6.8), and 5% \u0026beta;-mercaptoethanol (added fresh) and stored at \u0026minus;80 \u0026deg;C. RNA was extracted using the RNeasy Mini Kit (#74104, Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were assessed using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). Extracted RNA samples were stored at \u0026minus;80 \u0026deg;C prior to sequencing (n = 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 RNA Sequencing and Data Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-throughput RNA-Seq data were prepared using Lexogen Quantseq FWD kit for Illumina with polyA selection and sequenced on Illumina NovaSeq sequencer (run length 1x75 nt). Bcl files were converted to Fastq format using bcl2fastq v. 2.20.0.422 Illumina software for basecalling. Quality check of raw single-end fastq reads was carried out by FastQC [10]. The adapters and quality trimming of raw fastq reads was performed using Trimmomatic v0.39 [11] with settings CROP:250 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:5 MINLEN:35. Trimmed RNA-Seq reads were mapped against the mouse genome (mm38) and Ensembl GRCm38 v.93 annotation using STAR v2.7.3a [12] as splice-aware short read aligner and default parameters except --outFilterMismatchNoverLmax 0.66 and --twopassMode Basic. Quality control after alignment concerning the number and percentage of uniquely- and multi-mapped reads, rRNA contamination, mapped regions, read coverage distribution, strand specificity, gene biotypes and PCR duplication was performed using several tools namely RSeQC v4.0.0 [13], Picard toolkit v2.25.6 [14], Qualimap v.2.2.2 [15].\u003c/p\u003e\n\u003cp\u003eThe differential gene expression analysis was calculated based on the gene counts produced using featureCounts from Subread package v2.0 [16] and further analyzed by Bioconductor package DESeq2 v1.34.0 [17]. Data generated by DESeq2 with independent filtering were selected for the differential gene expression analysis due to its conservative features and to avoid potential false positive results. Genes were considered as differentially expressed based on a cut-off of adjusted p-value \u0026le; 0.05 and log2(fold-change) \u0026ge;1 or \u0026le;-1. Clustered heatmaps were generated from selected top differentially regulated genes using R package pheatmap v1.0.12 [18], volcano plots were produced using ggplot2 v3.3.5 package [19] and MA plots were genereted using ggpubr v0.4.0 package [20].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 RNA extraction, cDNA synthesis and reverse-transcription PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from mouse lacrimal glands or mLGECs and subsequently transcribed into cDNA as previously described \u003c!--[if supportFields]\u003e\u003cspan style='mso-element:field-begin'\u003e\u003c/span\u003e\u003cspan style='font-size:12.0pt;line-height:115%;color:windowtext'\u003eADDIN CitaviPlaceholder{{"$id":"1","$type":"SwissAcademic.Citavi.Citations.WordPlaceholder, SwissAcademic.Citavi","Entries":[{"$id":"2","$type":"SwissAcademic.Citavi.Citations.WordPlaceholderEntry, 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To verify cDNA integrity, \u0026beta;-actin PCR was performed on all samples. Each reaction was carried out using Taq DNA Polymerase (Thermo Fisher Scientific), 100 mM dNTPs (Thermo Fisher Scientific), and 2 \u0026times; 10 pmol gene-specific primers (Table 1), following the manufacturer\u0026rsquo;s instructions. The PCR products were then separated on a 2% agarose gel, stained with GelRed Nucleic Acid Gel Stain (Linaris, Dossenheim, Germany), and analyzed via electrophoresis alongside a Low Range DNA Ladder (#SM0383, Thermo Fisher Scientific).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Quantitative real-time PCR (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative real-time PCR analysis was performed using a LightCycler\u0026reg; 480 (Roche, Basel, Switzerland) in combination with Takyon\u0026trade; MasterMix (Eurogentec, Seraing, Belgium), following the manufacturer\u0026rsquo;s instructions. All reactions were conducted in triplicates, with a no-template control included. Primer efficiency was calculated for each primer used (Table 1), and data analysis was carried out according to Pfaffl \u003c!--[if supportFields]\u003e\u003cspan style='mso-element:field-begin'\u003e\u003c/span\u003e\u003cspan style='font-size:12.0pt; line-height:115%;color:windowtext'\u003eADDIN 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style='mso-element:field-separator'\u003e\u003c/span\u003e\u003c/span\u003e\u003c![endif]--\u003e[23]\u003c!--[if supportFields]\u003e\u003cspan style='font-size:12.0pt;line-height:115%;color:windowtext'\u003e\u003cspan style='mso-element:field-end'\u003e\u003c/span\u003e\u003c/span\u003e\u003c![endif]--\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Immunofluorescence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter two washes with PBS, cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes. Permeabilization was then performed using 0.1% Triton X, followed by additional PBS washes. The cells were subsequently incubated with 10% normal serum for one hour, then incubated overnight with the diluted primary antibody (Table 2). The following day, the cells were washed again with PBS and incubated for one hour with the secondary antibody (Table 2) in PBS. This was followed by a 5-minute incubation with DAPI (Sigma-Aldrich) and a final mounting using Fluorescence Mounting Medium (Agilent Technologies, Santa Clara, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 \u0026beta;-Hexosaminidase Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the secretory activity of mLGECs, the cells were stimulated with either 0.1 mM carbachol or 1 mM dibutyryl-cAMP (dbcAMP) in serum-free medium for 1 hour. Following stimulation, the supernatants were collected, the cells were lysed, and both the supernatants (SN) and lysates (L) were stored at \u0026ndash;80 \u0026deg;C until further analysis. \u0026beta;-hexosaminidase activity was measured using 4-methylumbelliferyl N-acetyl-\u0026beta;-D-glucosaminide (M2133, Sigma-Aldrich) as a substrate, and fluorescence was quantified at 450/365 nm (emission/excitation). The amount of \u0026beta;-hexosaminidase released was calculated as substrate turnover in the supernatant relative to the lysate, using the following formula:\u0026nbsp;beta-hexosaminidase release = (\u0026Delta;E\u003csub\u003eSN\u003c/sub\u003e * Factor\u003csub\u003eSN\u003c/sub\u003e)/(\u0026Delta;E\u003csub\u003eL\u003c/sub\u003e * Factor\u003csub\u003eL\u003c/sub\u003e) [24].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Ethanol (EtOH)\u003c/strong\u003e \u003cstrong\u003eInduced Damage of mLGECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emLGECs were left untreated or stimulated with 12.5 ng/mL recombinant mouse glial cell line-derived neurotrophic factor (GDNF; Thermo Fisher, #4504450UG), amphiregulin (AREG; Bio-Techne, #989-AR-100/CF), nerve growth factor (NGF; Bio-Techne, #1156-NG-100/CF), or heparin-binding epidermal growth factor-like growth factor (HBEGF; BIOZOL, #MCE-HY-P7194-50UG) for 2 hours, or treated with 10 nM SCH772984, an extracellular signal-regulated kinase (ERK) inhibitor (MedChemExpress, Monmouth Junction, USA) for 24 hours. Cells were then washed with PBS and exposed to 20% EtOH (v/v) for 1 minute. EtOH was aspirated, cells were washed with PBS, and fresh medium was added. Intracellular ATP levels were measured at 0, 1, 2, 4, 6, and 24 hour post-treatment. Samples for Annexin V/PI staining and RNA-seq were collected at 0, 2, and 6 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Intracellular ATP Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter stimulation mLGECs were washed with PBS and incubated with a 1:1 mixture of CellTiter-Glo\u0026reg; 3D Reagent (#G9683, Promega, Walldorf, Germany) and PBS for 10 minutes at room temperature. Luminescence was then measured using a CLARIOstar Plus plate reader (BMG Labtech, Ortenberg, Germany) according to the manufacturer\u0026apos;s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Annexin-V/PI for Flow Cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantification of life, dead, and apoptotic cells was performed using FITC-conjugated annexin\u0026nbsp;V and propidium iodide (PI) staining, followed by flow cytometric analysis. Treated and untreated mLGECs (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL) were collected, washed with PBS, and resuspended in 500 \u0026mu;L binding buffer. The cells were then stained with 10 \u0026mu;g/mL FITC-conjugated annexin V (#A13199, Thermo Fisher Scientific) and 50 \u0026mu;g/mL PI for 30 minutes at 4 \u0026deg;C in the dark. Samples were analyzed using a CytoFLEX S Flow Cytometer (Beckman Coulter, Krefeld, Germany), and data were processed using FlowJo software (FlowJo LLC; version 10.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 Wound Healing Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emLGECs were seeded at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL into a \u0026micro;-Dish 35 mm containing a 2-Well Culture-Insert (#81175, ibidi, Gr\u0026auml;felfing, Germany). After 24 hours of attachment, cells were stimulated with different growth factors (12.5 ng/mL; listed in Section 2.9, Ethanol (EtOH)-Induced Damage of mLGECs) for 2 hours, or with SCH772984 for 24 hours. Subsequently, the insert was removed, and the gap area was imaged at 0, 24, 48, and 72 hours using an inverted microscope (CKX53; Evident, Bad Kreuznach, Germany). Images were analyzed with cellSense Standard software version 4.2.1 (Evident).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.13 Enzyme-linked Immunosorbent Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor protein extraction, untreated and EtOH treated cells were detached using trypsin, washed with PBS, and lysed in Triton buffer (1% Triton X-100 in PBS) supplemented with protease and phosphatase inhibitors (2 \u0026micro;l/ml) for 30 minutes on ice. Total protein concentrations were determined using a BCA Protein Assay (#A55864, Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. Quantitative analysis of Hb-EGF and amphiregulin concentrations was performed using commercial ELISA kits (Mouse Amphiregulin DuoSet ELISA, #DY989; Mouse HB-EGF DuoSet ELISA, #DY8239-05; R\u0026amp;D Systems, Minneapolis, USA) following the protocols provided by the manufacturer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14 Statistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are presented as mean values with standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 10 (version 10.1.2, GraphPad Software Inc., San Diego, USA). Statistical significance was defined as a p-value of \u0026le; 0.05.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Transcriptomic profiling of mLGECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative analysis of the transcriptomic profiles of primary mLGECs (for isolation see \u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e) and native murine lacrimal gland tissue was conducted, which revealed a distinct difference in gene expression (\u003cstrong\u003eFigure 1A\u003c/strong\u003e). Among the top 20 differentially expressed genes, those associated with structural and contractile functions were strongly suppressed in mLGECs. Additionally, stress- and immune-related transcripts were prominently activated, together with genes involved in inflammatory, stress, and hormonal responses, as well as in metabolic processes (\u003cstrong\u003eSupplemental Table 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO) domain overrepresentation analysis supported these findings (\u003cstrong\u003eFigure 1B\u003c/strong\u003e). Among the upregulated categories, the most significantly enriched biological processes included the acute-phase response (Fold Enrichment 27.6, p = 4.7\u0026times;10⁻\u0026sup1;\u0026sup2;) and inflammatory response (6.0, p = 5.4\u0026times;10⁻\u0026sup1;\u0026sup2;). Enriched cellular components were dominated by the extracellular region (4.4, p = 2.0\u0026times;10⁻\u0026sup3;\u0026sup1;) and extracellular space (3.9, p = 1.2\u0026times;10⁻\u0026sup2;⁶), whereas enriched molecular functions included cytokine activity (9.0, p = 5.0\u0026times;10⁻\u0026sup1;⁴) and growth factor activity (10.8, p = 4.3\u0026times;10⁻\u0026sup1;\u0026sup2;). In contrast, downregulated categories were dominated by contractile and muscle-associated terms. Biological processes included muscle contraction (33.1, p = 3.7\u0026times;10⁻\u0026sup2;⁰) and sarcomere organization (28.9, p = 3.2\u0026times;10⁻\u0026sup1;\u0026sup2;). Cellular components such as the Z-disc (14.1, p = 1.1\u0026times;10⁻\u0026sup1;⁴) and I-band (38.4, p = 5.1\u0026times;10⁻\u0026sup1;\u0026sup1;) were significantly reduced. Molecular functions associated with contractility, including actin filament binding (6.4, p = 2.9\u0026times;10⁻⁷) and actin binding (29.7, p = 1.4\u0026times;10⁻⁶), were likewise suppressed.\u003c/p\u003e\n\u003cp\u003eUsing a transcriptome atlas of the mouse lacrimal gland, a more detailed examination of stromal and epithelial subpopulations was conducted by averaging log2 fold-changes across their marker genes (unweighted mean per subgroup) (\u003cstrong\u003eFigure 1C\u003c/strong\u003e). All cell type populations were reduced in cultured mLGECs compared with native tissue, including fibroblasts (\u0026minus;1.43), endothelial (\u0026minus;6.49), mural (\u0026minus;1.07), lymphoid (\u0026minus;1.15), and myeloid cells (\u0026minus;2.73). Similarly, epithelial clusters such as acinar (\u0026minus;1.54), ductal (\u0026minus;2.30), and myoepithelial cells (\u0026minus;1.70) showed global downregulation. These results indicate a uniform loss of stromal and epithelial programs, consistent with dedifferentiation during adaptation to \u003cem\u003ein vitro\u003c/em\u003e culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Expression of epithelial and gland-specific markers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEpithelial and lacrimal gland specific markers were assessed across passages P1 to P3 by RT-PCR, using native lacrimal gland tissue (mLG) and 3T3 fibroblasts as controls, and additionally by immunocytochemistry at P1 (\u003cstrong\u003eFigure 2A, B\u003c/strong\u003e). Bands were detected for \u003cem\u003eAqp5\u003c/em\u003e (144 bp), \u003cem\u003eRab3d\u003c/em\u003e (130 bp), \u003cem\u003eLyz1\u003c/em\u003e (100 bp), \u003cem\u003eLtf\u003c/em\u003e (191 bp), \u003cem\u003eKrt5\u003c/em\u003e (177 bp), \u003cem\u003eFoxc1\u003c/em\u003e (171 bp), \u003cem\u003eSox10\u003c/em\u003e (147 bp), \u003cem\u003ePax6\u003c/em\u003e (245 bp), \u003cem\u003eActa2\u003c/em\u003e (211 bp), and \u003cem\u003eVim\u003c/em\u003e (327 bp) across passages P1\u0026ndash;P3 and native lacrimal gland tissue, while \u003cem\u003eLtf\u003c/em\u003e and \u003cem\u003ePax6\u003c/em\u003e were absent in 3T3 cells (Figure 2A). Semi-quantitative analysis normalized to \u003cem\u003eActb\u003c/em\u003e indicated a slight decrease of epithelial markers from P1 to P3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtein-level validation by immunocytochemistry showed membrane-associated AQP5 staining, cytoplasmic granular staining for LTF, cytoplasmic KRT5 staining, nuclear FOXC1/2, and \u0026alpha;-SMA was detected in the cytoplasm. VIM staining was absent in mLGECs but strongly expressed in 3T3 fibroblasts (\u003cstrong\u003eFigure 2B\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunctional analysis of secretory activity demonstrated that \u0026beta;-hexosaminidase release increased upon stimulation. Both carbachol (mean difference \u0026minus;0.1417, p = 0.0066) and dibutyryl cAMP (db-cAMP) (mean difference \u0026minus;0.1847, p = 0.0004) induced significantly higher secretion compared to controls (\u003cstrong\u003eFigure 2C\u003c/strong\u003e), confirming at least a partial preservation of regulated exocytosis and secretory capacity in cultured mLGECs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Acute injury and early regeneration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcute injury was induced by exposing mLGECs to 20% ethanol for 1 minute, followed by incubation in either serum-free or serum-containing medium (\u003cstrong\u003eFigure 3A\u003c/strong\u003e). Cell viability was assessed immediately prior to ethanol exposure and at 0, 1, 2, 4, and 24 hours post-treatment. A significant decline of viability to 40.95 \u0026plusmn; 16.56% to the baseline value (p \u0026lt; 0.0001) was observed immediately after exposure, followed by partial recovery at 1 hour (55.04 \u0026plusmn; 6.09%) and further increase at 2 hours (72.49 \u0026plusmn; 11.54%) under serum conditions. At 48 hours (data not shown), viability exceeded baseline levels (24 h: 171.30 \u0026plusmn; 39.96%; 48 h: 287.80 \u0026plusmn; 116.28%), with minimal differences between treated and untreated cells, indicating full recovery. As serum withdrawal reduced growth in untreated controls, subsequent experiments were performed in serum-containing medium (\u003cstrong\u003eFigure 3B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eApoptosis was assessed by Annexin V and propidium iodide staining to validate regulated cell death as the driving force behind reduced cell viability. Immediately after ethanol exposure, the proportion of living cells decreased significantly (p \u0026lt; 0.0001) with a concomitant increase in dead cells (p \u0026lt; 0.0001). At this point no apoptotic cells were detected and probably other forms of cell death occur. \u0026nbsp;At 2 hours, apoptotic cells were significantly elevated (p \u0026lt; 0.0001) while the fraction of living cells further declined (p = 0.0065). By 6 hours, the distribution of live, apoptotic, and dead cells returned to baseline levels, indicating a consistent recovery trend. This kinetics mirrored the ATP-based viability measurements, showing an early decline followed by gradual recovery (\u003cstrong\u003eFigure 3C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo gain an unbiased insight into pathways that could induce cell recovery and be protective for mLGECs, we performed total mRNA sequencing at the 2-hour time point in serum-containing medium. This timepoint was selected, as it represents the early recovery phase, which is characterized by partial restoration of viability and the peak in apoptotic signaling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Transcriptomic analysis of intrinsic regeneration mechanisms after acute injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal mRNA sequencing revealed that among the top 20 differentially expressed genes, stress- and autophagy-related genes were strongly upregulated together with immune and interferon-associated transcripts. In contrast, a developmental regulator was downregulated (\u003cstrong\u003eFigure 4A, Supplemental Table 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eGO overrepresentation analysis of upregulated genes highlighted significantly enriched biological processes, including leukocyte cell\u0026ndash;cell adhesion (Fold Enrichment 14.2, p = 1.1 \u0026times; 10⁻⁶) and response to virus (6.1, p = 1.3 \u0026times; 10⁻⁵). The top enriched cellular components were extracellular region (Fold Enrichment 1.8, p = 9.8 \u0026times; 10⁻⁶) and cell projection (1.9, p = 1.9 \u0026times; 10⁻⁴). At the molecular function level, significant terms comprised sequence-specific double-stranded DNA binding (Fold Enrichment 2.6, p = 2.4 \u0026times; 10⁻⁵) and receptor agonist activity (6.3, p = 8.5 \u0026times; 10⁻⁵). KEGG pathway analysis revealed enrichment of MAPK signaling (2.3, p = 4.3\u0026times;10⁻⁴), TNF signaling (3.1, p = 1.5 \u0026times; 10⁻\u0026sup3;) and IL-17 signaling (3.2, p = 3.6 \u0026times; 10⁻\u0026sup3;) (\u003cstrong\u003eFigure 4B, top\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eDownregulated categories were dominated by transcription- and development-related processes. The most significant biological processes were regulation of transcription from RNA polymerase II promoter (Fold Enrichment 4.5, p = 9.1 \u0026times; 10⁻⁴) and positive regulation of endothelial cell migration (43.8, p = 1.9 \u0026times; 10⁻\u0026sup3;). At the molecular function level, significantly reduced terms included RNA polymerase II transcription factor activity, sequence-specific DNA binding (Fold Enrichment 5.7, p = 2.3 \u0026times; 10⁻⁴) and growth factor activity (16.2, p = 1.3 \u0026times; 10⁻\u0026sup2;). (\u003cstrong\u003eFigure 4B, bottom\u003c/strong\u003e). Based on this analysis, TNF and MAPK signaling were selected for detailed follow-up analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 MAPK and TNF Signaling Pathways: Regeneration Mechanisms After Acute Injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcute injury activated both TNFR1 and TNFR2-mediated arms of TNF signaling, with increased expression across adaptor and kinase nodes and their immediate-early outputs. Downstream effectors of inflammation and survival were induced, including the \u003cem\u003eFos/Jun\u003c/em\u003e family (\u003cem\u003eFos\u003c/em\u003e log2FC 2.87, p = 3.9\u0026times;10⁻⁵; \u003cem\u003eJun\u003c/em\u003e 2.26, p = 1.5\u0026times;10⁻⁸), NF-\u0026kappa;B regulators (\u003cem\u003eRela\u003c/em\u003e 1.45, p = 3.7\u0026times;10⁻⁵\u0026sup2;; \u003cem\u003eNfkbia\u003c/em\u003e 1.10, p = 3.9\u0026times;10⁻⁹), inflammatory cytokines (\u003cem\u003eTnf\u003c/em\u003e 2.22, p = 0.013), chemokines involved in leukocyte recruitment (\u003cem\u003eCcl5\u003c/em\u003e 4.94, p = 4.3\u0026times;10⁻⁷⁸; \u003cem\u003eCxcl10\u003c/em\u003e 4.75, p = 4.7\u0026times;10⁻⁵⁵; \u003cem\u003eCxcl2\u003c/em\u003e 2.78, p = 1.4\u0026times;10⁻⁶), and adhesion/vascular genes (\u003cem\u003eIcam1\u003c/em\u003e 1.38, p = 1.1\u0026times;10⁻⁷; \u003cem\u003eSele\u003c/em\u003e 2.06, p = 0.048; \u003cem\u003eEdn2\u003c/em\u003e 2.14, p = 0.0044). Additional immune regulators were upregulated, including \u003cem\u003eSqstm1\u003c/em\u003e (1.68, p = 5.1\u0026times;10⁻⁶⁹), \u003cem\u003eTraf1\u003c/em\u003e (1.62, p = 0.045), and \u003cem\u003eIl18r1\u003c/em\u003e (4.33, p = 1.3\u0026times;10⁻⁶).\u003c/p\u003e\n\u003cp\u003eIn addition to \u003cem\u003eTnf\u003c/em\u003e, multiple growth factor-receptor-associated ligands of the MAPK pathway were differentially expressed. Significant upregulation was observed for \u003cem\u003eHbegf\u003c/em\u003e (2.69, p = 9.2\u0026times;10⁻⁶⁶), \u003cem\u003eAreg\u003c/em\u003e (2.95, p = 1.1\u0026times;10⁻\u0026sup2;\u0026sup2;), \u003cem\u003eNgf\u003c/em\u003e (2.88, p = 4.9\u0026times;10⁻⁴\u0026sup3;), \u003cem\u003eGdnf\u003c/em\u003e (3.16, p = 5.2\u0026times;10⁻\u0026sup2;\u0026sup1;), \u003cem\u003eEreg\u003c/em\u003e (1.06, p = 2.6\u0026times;10⁻⁹), \u003cem\u003eFgf9\u003c/em\u003e (2.18, p = 0.0046), \u003cem\u003eEfna1\u003c/em\u003e (1.31, p = 2.9\u0026times;10⁻\u0026sup1;\u0026sup1;), \u003cem\u003eAngpt2\u003c/em\u003e (1.20, p = 0.045), \u003cem\u003ePdgfa\u003c/em\u003e (1.25, p = 2.8\u0026times;10⁻⁴\u0026sup3;), \u003cem\u003eKitl\u003c/em\u003e (1.14, p = 3.3\u0026times;10⁻\u0026sup2;⁵), \u003cem\u003eEpha2\u003c/em\u003e (1.61, p = 1.3\u0026times;10⁻⁵), \u003cem\u003eKdr\u003c/em\u003e (1.48, p = 3.3\u0026times;10⁻⁴), and \u003cem\u003eMet\u003c/em\u003e (1.53, p = 2.0\u0026times;10⁻\u0026sup1;⁴). In contrast, \u003cem\u003eFgf2\u003c/em\u003e (\u0026ndash;2.19, p = 9.9\u0026times;10⁻\u0026sup2;⁸), \u003cem\u003eFgf10\u003c/em\u003e (\u0026ndash;1.07, p = 9.0\u0026times;10⁻⁹), and \u003cem\u003eEgf\u003c/em\u003e (\u0026ndash;2.31, p = 1.4\u0026times;10⁻⁶) were significantly downregulated. Furthermore, the cascade elements and downstream transcriptional targets were also affected. Significant upregulation was observed for \u003cem\u003eKit\u003c/em\u003e (2.50, p = 8.0\u0026times;10⁻⁴), \u003cem\u003eMknk2\u003c/em\u003e (1.31, p = 1.2\u0026times;10⁻\u0026sup2;⁹), \u003cem\u003eAtf3\u003c/em\u003e (5.54, p = 3.8\u0026times;10⁻\u0026sup3;⁹), \u003cem\u003eCreb5\u003c/em\u003e (2.01, p = 8.3\u0026times;10⁻\u0026sup2;⁸), \u003cem\u003eRasgrf1\u003c/em\u003e (2.36, p = 0.0084), and \u003cem\u003eCacna2d2\u003c/em\u003e (2.75, p = 0.0019)\u003cstrong\u003e\u0026nbsp;(Figure 5A, B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo validate differential MAPK-pathway regulation after acute injury, the expression of selected components was quantified by qPCR. Genes encoding signaling ligands (\u003cem\u003eHbegf, Areg, Fgf9, Gdnf, Ngf, Efna1, Egf\u003c/em\u003e), cascade elements (\u003cem\u003eKit, Epha2\u003c/em\u003e), and downstream targets (\u003cem\u003eCreb5, Jun, Fos\u003c/em\u003e) were examined. Two hours after injury, all genes except \u003cem\u003eFgf9, Efna1,\u003c/em\u003e and \u003cem\u003eKit\u003c/em\u003e were significantly altered and \u003cem\u003eEfna1\u003c/em\u003e and \u003cem\u003eKit\u003c/em\u003e showed a tendency toward upregulation (Figure 5C). Comparing mean normalized expression (MNE) in untreated samples revealed relatively high basal levels of \u003cem\u003eEpha2\u003c/em\u003e (1.6\u0026times;10⁻\u0026sup1; \u0026plusmn; 3.3\u0026times;10⁻\u0026sup2;), \u003cem\u003eHbegf\u003c/em\u003e (6.3\u0026times;10⁻\u0026sup2; \u0026plusmn; 2.5\u0026times;10⁻\u0026sup2;), and \u003cem\u003eNgf\u003c/em\u003e (2.9\u0026times;10⁻\u0026sup2; \u0026plusmn; 7.0\u0026times;10⁻\u0026sup3;), whereas \u003cem\u003eAreg\u003c/em\u003e (1.4\u0026times;10⁻⁵ \u0026plusmn; 6.3\u0026times;10⁻⁶) and \u003cem\u003eKit\u003c/em\u003e (5.4\u0026times;10⁻⁶ \u0026plusmn; 1.4\u0026times;10⁻⁶) exhibited low baseline expression (\u003cstrong\u003eSupplementary Figure 2A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFor all confirmed significant signaling ligands, expression was further profiled over a 24-hour period following acute ethanol injury. \u003cem\u003eHbegf\u003c/em\u003e was strongly induced at 2 hours (p = 0.0008) and peaked at 4 hours (p \u0026lt; 0.0001). \u003cem\u003eAreg\u003c/em\u003e was significantly upregulated at 2 hours (p = 0.0007) and 4 hours (p = 0.0016). \u003cem\u003eGdnf\u003c/em\u003e showed significant elevation at 4 hours (p \u0026lt; 0.0001), and \u003cem\u003eNgf\u003c/em\u003e peaked at 4 hours (p \u0026lt; 0.0001). In contrast, \u003cem\u003eEgf\u003c/em\u003e was significantly downregulated at 2 hours (p \u0026lt; 0.0001), with a weaker but still detectable reduction at 4 hours (p = 0.037) (\u003cstrong\u003eSupplementary Figure 2B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Growth Factor\u0026ndash;Mediated Regeneration of mLGECs After Acute Injury\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate whether MAPK growth factors contribute to regeneration after ethanol-induced injury, we performed cell viability and wound-healing assays. Dose\u0026ndash;response analyses confirmed high-affinity activity for HBEGF, NGF, GDNF, and AREG (Kd \u0026lt; 1 ng/ml, saturation at 12.5 ng/ml) and identified a non-cytotoxic concentration of 10 nM for the MAPK inhibitor SCH772984 (IC₅₀ 32.7\u0026ndash;98.5 nM across donors) (\u003cstrong\u003eFigure 5A, Supplementary Figure 2C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFollowing ethanol exposure, all MAPK growth factors enhanced viability recovery, whereas SCH772984 caused a progressive decline (\u003cstrong\u003eFigure 6A, Supplementary Figure 3\u003c/strong\u003e). Viability decreased by more than 50% at 0 hours under all conditions, excluding acute protective effects. AREG showed the earliest improvement at 2 hours (p = 0.0217). By 4 hours, all factors significantly promoted recovery (p \u0026lt; 0.01) and by 24 hours, HBEGF exerted the strongest effect (+66%, p \u0026lt; 0.0001), followed by AREG (+44%), GDNF (+22%), and NGF (+15%). Pairwise comparisons confirmed differential efficacy, with HBEGF outperforming GDNF (p = 0.0004) and NGF (p \u0026lt; 0.0001), and AREG showing greater activity than NGF (p = 0.0140).\u003c/p\u003e\n\u003cp\u003eWound-healing assays further substantiated these findings. \u003cstrong\u003eAll MAPK growth factors accelerated wound closure relative to untreated controls\u003c/strong\u003e (p \u0026lt; 0.0001), whereas SCH772984 markedly impaired closure (\u0026minus;38%, p \u0026lt; 0.0001) (Figure 5B). Among growth factors,\u003cstrong\u003e\u0026nbsp;HBEGF was significantly more effective than GDNF (+30%, p = 0.0009) and NGF (+27%, p = 0.0035)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eFigure 6B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFinally, protein-level analyses aligned with transcriptional findings. Both HBEGF and AREG were transiently upregulated within the first two hours after ethanol exposure before returning to baseline, paralleling their mRNA profiles. HBEGF showed a robust peak at 2 hours (p = 0.0003), while AREG exhibited a weaker but significant induction at the same time point (p = 0.027). Across all time points, HBEGF levels remained consistently higher than those of AREG, consistent with gene-expression profiles (\u003cstrong\u003eFigure 6C\u003c/strong\u003e).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Characterization of Acinar Epithelial Cells from Murine Lacrimal Glands (mLGEC)\u003c/h2\u003e\u003cp\u003eTranscriptomic profiling of mLGECs revealed a pronounced downregulation of contractile programs. This could reflect skeletal muscle contamination during dissection due to the gland\u0026rsquo;s anatomical proximity to the masseter muscle [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] or selective vulnerability of myoepithelial cells during enzymatic isolation and culture [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Nevertheless, the persistence of \u003cem\u003eActa2/α-SMA\u003c/em\u003e expression suggests that myoepithelial cells are not completely absent, but rather that contractile pathways are broadly suppressed under \u003cem\u003ein vitro\u003c/em\u003e conditions.\u003c/p\u003e\u003cp\u003eIn contrast to the loss of contractile signatures, stress- and immune-related genes were strongly upregulated in cultured mLGECs. Among the genes, \u003cem\u003eSgk1\u003c/em\u003e, encoding for a well-characterized stress-responsive kinase, has been shown to be rapidly induced under diverse cell-stress conditions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Similarly, increased expression of \u003cem\u003ePtx3\u003c/em\u003e, \u003cem\u003eCxcl5\u003c/em\u003e, \u003cem\u003eSpp1\u003c/em\u003e, and \u003cem\u003eSerpine1\u003c/em\u003e suggests enhanced immunomodulatory activity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The induction of \u003cem\u003eCyp1b1\u003c/em\u003e is consistent with metabolic adaptation and survival mechanisms, as CYP1B1 has been shown to promote proliferation, inhibit apoptosis, and facilitate cell-cycle progression during \u003cem\u003ein vitro\u003c/em\u003e differentiation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Collectively, these findings indicate that mLGECs engage a broad stress- and immune-related program as part of their adaptation process, likely in response to the absence of physiological signaling cues and exposure to artificial medium components [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUsing the mouse lacrimal gland transcriptome atlas [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], we examined marker genes for stromal populations and epithelial cell clusters. Stromal cells included fibroblasts that form the interstitial connective tissue; vascular cells comprising endothelial and mural cell populations across venous, arterial, and capillary vessels [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; and resident immune cells of lymphoid and myeloid lineages that mediate immune surveillance and defense within the interstitium [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Epithelial clusters comprised of acinar cells secreting aqueous tear components, ductal epithelial cells forming the excretory duct system, and myoepithelial cells providing contractile support to acini. Notably, both stromal and epithelial subpopulations exhibited global downregulation in culture, a pattern consistent with partial dedifferentiation, a well‑recognized phenomenon following cell isolation and adaptation to two‑dimensional culture systems [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite the global transcriptomic shift observed in culture, mLGECs retained key epithelial and lacrimal gland specific features. Core functional markers, including \u003cem\u003eAqp5\u003c/em\u003e and \u003cem\u003eRab3d\u003c/em\u003e, essential for water transport and vesicle trafficking [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], as well as secretory proteins \u003cem\u003eLtf\u003c/em\u003e and \u003cem\u003eLyz1\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], remained expressed, with AQP5 and LTF confirmed at the protein level. The basal epithelial identity was further supported by \u003cem\u003eKrt5\u003c/em\u003e expression, while developmental regulators \u003cem\u003eFoxc1\u003c/em\u003e, \u003cem\u003eSox10\u003c/em\u003e, and \u003cem\u003ePax6\u003c/em\u003e [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] were preserved, albeit with reduced \u003cem\u003ePax6\u003c/em\u003e levels compared to native tissue. Notably, the absence of VIM protein in mLGECs, despite transcript detection, contrasted with its strong expression in 3T3 fibroblasts arguing against fibroblast contamination.\u003c/p\u003e\u003cp\u003eFunctionally, mLGECs retained regulated exocytosis, as demonstrated by β-hexosaminidase release, indicating preserved secretory function despite partial dedifferentiation and stress adaptation. Collectively, these findings indicate that while culture conditions induce transcriptional remodeling, mLGECs retain key hallmarks of lacrimal gland epithelial identity and maintain their functionality during early passages.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Acute Injury Responses and Intrinsic Regeneration Mechanisms\u003c/h2\u003e\u003cp\u003eOur findings demonstrate that ethanol exposure induces a rapid injury response in mLGECs, followed by early signs of recovery. Ethanol is widely used as a nonspecific cytotoxic agent, as it partitions into lipid bilayers, increases membrane fluidity and permeability, disrupts barrier properties, and denatures proteins, collectively causing ATP depletion and apoptotic or lytic cell death in epithelial cells [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The sharp decline in viability and increase in apoptosis within the first hours after exposure mirror injury kinetics reported in corneal epithelial cells [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], confirming ethanol as a robust model of acute-injury. Importantly, recovery dynamics within 24\u0026ndash;48 hours, even under serum-free conditions, suggest that mLGECs have an inherent intrinsic regenerative capacity as seen \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This resilience likely reflects activation of conserved stress-response and repair programs that support epithelial survival after acute cytotoxic insults [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTranscriptomic profiling reinforced this interpretation, revealing strong induction of stress-, immune-, and repair-associated pathways alongside suppression of transcriptional and developmental programs. Upregulation of canonical stress regulators such as \u003cem\u003eSqstm1\u003c/em\u003e, \u003cem\u003eFnip1\u003c/em\u003e, and \u003cem\u003eNdrg1\u003c/em\u003e together with GO biological-process enrichment for response to virus, inflammatory response, and leukocyte cell\u0026ndash;cell adhesion suggests the induction of autophagy, metabolic adaptation, and energy-sensing pathways. This is consistent with reports that epithelial cells engage conserved stress responses to maintain survival after injury [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Concurrent induction of chemokines like \u003cem\u003eCcl5\u003c/em\u003e and interferon-stimulated genes such as \u003cem\u003eIfit1\u003c/em\u003e, along with enrichment for the GO cellular components cytoplasm, cell projection, and axoneme, reflects early immunomodulatory responses, aligning with studies showing rapid cytokine release and immune activation in ocular epithelia following acute insults [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Importantly, the expression of repair-associated factors, including \u003cem\u003eKlf5\u003c/em\u003e and \u003cem\u003eHbegf\u003c/em\u003e, highlights that regenerative mechanisms are engaged already during the early recovery phase, consistent with evidence that growth-factor signaling via EGFR ligands promotes epithelial wound healing [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This pattern mirrors findings from animal models of lacrimal gland injury, which report immediate inflammatory and immune responses, including early upregulation of Il1b (IL-1β) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The downregulation of \u003cem\u003eId3\u003c/em\u003e and associated transcriptional regulators suggests a transient suppression of differentiation and developmental processes [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], likely reflecting a trade-off between energy-demanding transcriptional activity and immediate stress adaptation [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], aligning with GO enrichment analyses showing reduced transcription-related processes.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTogether, these observations support a model in which acute ethanol injury elicits a tightly coordinated early response program in mLGECs. Rapid stress adaptation and innate immune activation, which occur alongside the prompt engagement of intrinsic repair pathways and the transient recession of differentiation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Growth Factor\u0026ndash;Driven Regeneration in the Injured Lacrimal Gland\u003c/h2\u003e\u003cp\u003eAcute epithelial injury to mlGECs rapidly activates both TNF and MAPK signaling, underscoring the interplay of inflammatory and regenerative mechanisms in the early repair phase. Upregulation of TNFR1- and TNFR2-associated transcripts, along with downstream effectors such as \u003cem\u003eFos\u003c/em\u003e, \u003cem\u003eJun\u003c/em\u003e, NF-κB regulators, cytokines (\u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eIl6\u003c/em\u003e, \u003cem\u003eTnf\u003c/em\u003e), chemokines (\u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eCcl5\u003c/em\u003e, \u003cem\u003eCxcl2\u003c/em\u003e) and adhesion molecules (\u003cem\u003eIcam1\u003c/em\u003e, \u003cem\u003eSele\u003c/em\u003e, \u003cem\u003eVcam1\u003c/em\u003e, \u003cem\u003eVegfa\u003c/em\u003e, \u003cem\u003eEdn\u003c/em\u003e), reflects a \u0026ldquo;damage-control\u0026rdquo; response that promotes rapid immune-cell recruitment, vascular stabilization, and barrier protection while loosening the matrix to facilitate repair [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Although TNF signaling clearly contributes to regeneration, its strong proinflammatory and cytotoxic properties limits its suitability as a therapeutic target [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast, the MAPK pathway emerged as a key driver of epithelial regeneration. Multiple growth-associated ligands were differentially regulated, including upregulation of \u003cem\u003eHbegf\u003c/em\u003e, \u003cem\u003eAreg\u003c/em\u003e, \u003cem\u003eNgf\u003c/em\u003e, \u003cem\u003eGdnf\u003c/em\u003e, \u003cem\u003eFgf9\u003c/em\u003e, and \u003cem\u003eEreg\u003c/em\u003e, while \u003cem\u003eFgf2\u003c/em\u003e, \u003cem\u003eFgf10\u003c/em\u003e, and \u003cem\u003eEgf\u003c/em\u003e were downregulated. EGF-like factors such as HBEGF, AREG, EREG, and EGF typically promote cell proliferation and tissue renewal and can selectively modulate lacrimal gland functions [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Notably, \u003cem\u003eEgf\u003c/em\u003e expression declined following ethanol injury, despite exogenous EGF in the culture medium supporting cell survival and, at higher concentrations, enhancing regeneration. This reduction likely reflects negative feedback regulation of endogenous \u003cem\u003eEgf\u003c/em\u003e in the presence of extracellular ligand, a well-characterized mechanism in EGFR signaling [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Thus, while mLGECs suppress their own \u003cem\u003eEgf\u003c/em\u003e production, EGFR remains responsive, and regeneration appears to be preferentially driven by inducible ligands such as HBEGF and AREG [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to the EGF family, FGF family members, including \u003cem\u003eFGF2\u003c/em\u003e, \u003cem\u003eFGF9\u003c/em\u003e, and \u003cem\u003eFGF10\u003c/em\u003e, regulate cell growth, differentiation, and development. In the lacrimal gland, mesenchymal FGF10 signaling via epithelial FGFR2b drives gland induction and branching during development [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] and, supports epithelial proliferation in adult or regenerative contexts [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The observed downregulation of Fgf10 following EtOH injury likely reflects stage-specific dynamics, with early injury and inflammatory responses preceding later regenerative proliferation, and does not contradict its established role [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Neurotrophic factors, including NGF and GDNF promote cell survival and regeneration, particularly in neuronal and epithelial tissues. In the lacrimal gland, NGF likely functions in an autocrine/paracrine manner to maintain epithelial function and secretion [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In parallel, the GDNF-family ligand neurturin is critical for parasympathetic innervation of the lacrimal gland: neurturin-deficient (NRTN⁻/⁻) mice display markedly reduced parasympathetic nerve fibers, diminished tear production and a DED phenotype [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTaken together, these findings position MAPK signaling as a central regulator of lacrimal gland homeostasis, stress adaptation, and regeneration. Consistent with this, modulation of the p38 and JNK pathways in models of radiation-induced lacrimal gland damage markedly reduced tissue damage and improved glandular function, underscoring the importance of MAPK signaling in tissue repair [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Our data extend these findings by demonstrating that MAPK signaling is not only intrinsically activated in lacrimal gland cells following acute injury but can also be further enhanced by stimulation with HBEGF, AREG, NGF, GDNF and EGF. Although no acute protective effects were observed, enhanced regenerative recovery was evident, with HBEGF and AREG exerting the strongest effects. Importantly, pharmacological MAPK inhibition abrogated recovery, highlighting the essential role of this pathway in epithelial repair.\u003c/p\u003e\u003cp\u003eClinically, these results align with regenerative therapies already employed in patients with severe DED. Autologous serum (AS) eye drops, enriched in growth factors such as EGF, TGF-β, NGF, IGF, and PDGF, are widely used and improve tear stability, corneal epithelial healing, and patient symptoms, particularly in Sjogren\u0026rsquo;s syndrome and ocular surface disorders [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Similarly, platelet-rich plasma (PRP) offers higher concentrations of platelets and growth factors than AS and can be administered via direct injection into the human lacrimal gland. Recent clinical studies demonstrated that intraglandular PRP injections significantly enhanced tear secretion, reduced ocular surface damage, and alleviated symptoms in severe DED, highlighting their regenerative potential at the glandular level [\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. However, both AS and PRP are limited by donor variability, labor-intensive preparation, risks of contamination or instability, and complex regulatory classification as blood-derived biologics, restricting broader clinical application [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese limitations have sparked growing interest in replacing blood-derived products with standardized cocktails of defined growth factors, aiming for greater consistent efficacy and safety. Comparative studies emphasize that the regenerative activity of PRP largely depends on its content of factors like EGF, NGF, IGF, and PDGF, underscoring the need to identify the key components and their optimal concentrations [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Moreover, recent perspectives explicitly call for the development of controlled and optimized PRP-derived formulations, highlighting the need for standardization or substitution with defined growth factor combinations [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. In this context, our study provides critical insight by revealing growth factors intrinsically upregulated following acute injury in isolated mLGECs, offering a rational framework for designing targeted therapeutic cocktails to enhance lacrimal gland regeneration. Although pan-MAPK activation is undesirable due to its association with tumorigenesis and inflammation [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], a transient and pathway-specific stimulation could potentially promote lacrimal gland self-renewal.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study demonstrates that murine lacrimal gland acinar epithelial cells (mLGECs) undergo transcriptional remodeling in culture, characterized by downregulation of contractile programs and partial dedifferentiation, while retaining key lacrimal gland and epithelial markers and preserving their secretory capacity. Acute ethanol injury triggered a rapid stress and inflammatory response, including induction of \u003cem\u003eFos\u003c/em\u003e, \u003cem\u003eJun\u003c/em\u003e, NF-κB regulators, cytokines, and chemokines, reflecting a damage-control phase that stabilizes the tissue and prepares it for repair. Simultaneously, regenerative pathways were activated, with strong upregulation of MAPK ligands such as \u003cem\u003eHbegf\u003c/em\u003e, \u003cem\u003eAreg\u003c/em\u003e, \u003cem\u003eNgf\u003c/em\u003e, and \u003cem\u003eGdnf\u003c/em\u003e. Functional assays identified HBEGF and AREG as the most potent mediators of recovery, while MAPK inhibition completely abolished regeneration, underscoring the essential role of this pathway. Clinically, these findings align with AS and PRP therapies in severe DED, whose efficacy depends on growth factor content but is limited by its variability, preparation complexity, and regulatory challenges. By defining the intrinsically induced growth factors during lacrimal gland epithelial acinar cell regeneration after acute injury, our study establishes a rational preclinical basis on the road to developing standardized, safe, and effective growth factor-based therapies, opening a viable alternative to blood-derived products and new opportunities for targeted regenerative strategies in human lacrimal gland injury and disease in the future.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eADDE, Aqueous-Deficient Dry Eye; AQP5, Aquaporin 5; AREG, Amphiregulin; ATP, Adenosine Triphosphate; cAMP, Cyclic Adenosine Monophosphate; db-cAMP, Dibutyryl Cyclic Adenosine Monophosphate; DED, Dry Eye Disease; DMEM, Dulbecco\u0026rsquo;s Modified Eagle Medium; EGF, Epidermal Growth Factor; ELISA, Enzyme-Linked Immunosorbent Assay; EtOH, Ethanol; FCS, Fetal Calf Serum; FGF, Fibroblast Growth Factor; FOXC1/2, Forkhead Box Protein C1/C2; GDNF, Glial Cell Line-Derived Neurotrophic Factor; GO, Gene Ontology; HB-EGF (HBEGF), Heparin-Binding EGF-Like Growth Factor; IL, Interleukin; KEGG, Kyoto Encyclopedia of Genes and Genomes; KRT5, Keratin 5; LG, Lacrimal Gland; LTF, Lactotransferrin; LYZ1, Lysozyme 1; MAPK, Mitogen-Activated Protein Kinase; mLG, Murine Lacrimal Gland; mLGECs, Murine Lacrimal Gland Epithelial Cells; mRNA, Messenger Ribonucleic Acid; NF-\u0026kappa;B, Nuclear Factor Kappa B; NGF, Nerve Growth Factor; PAX6, Paired Box 6; PBS, Phosphate-Buffered Saline; PCR, Polymerase Chain Reaction; PI, Propidium Iodide; PRP, Platelet-Rich Plasma; qPCR, Quantitative Polymerase Chain Reaction; RNA-seq, RNA Sequencing; RT-PCR, Reverse Transcription Polymerase Chain Reaction; SCH772984, Selective MAPK/ERK Kinase Inhibitor; SOX10, SRY-Box Transcription Factor 10; TNF, Tumor Necrosis Factor; TNFR, Tumor Necrosis Factor Receptor; VIM, Vimentin\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFinancial Disclosure \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no proprietary or commercial interest in any materials discussed in this manuscript. FP receives royalties from Elsevier for the 25\u003csup\u003eth\u003c/sup\u003e Ed. of the anatomy atlas \u0026ldquo;Sobotta\u0026rdquo; and FP for the 3\u003csup\u003erd\u003c/sup\u003e of the \u0026ldquo;Sobotta Textbook of Anatomy\u0026rdquo;. The present work was performed in fulfillment of the requirements of obtaining the degree Dr. rer. nat. (IZ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by: Ernst und Berta Grimmke Foundation (project 6/21) to JD; in part by the German Research Foundation (DFG, grants PA738/15-1 and PA738/15-2) to FP; by a Sicca-Research-Award (Sicca-F\u0026ouml;rderpreis) of the Dry Eye and Ocular Surface Disease Resort of the Professional Association of German Ophthalmologists to IZ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the valuable support provided by Maike Hemmerlein, Anke Fischer-G\u0026ouml;\u0026szlig;wein (molecular biology), Hong Nguyen (immunohistochemistry) and J\u0026ouml;rg Pekarsky (graphics). Core Facility Genomics and Core Facility Bioinformatics of CEITEC Masaryk University are gratefully acknowledged for their support with obtaining scientific data presented in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: I.Z., J.D. and P.A.; methodology: I.Z.; validation: I.Z.; formal analysis: I.Z., N.B. and P.A.; investigation: I.Z. and H.F.; resources: P.A. and F.P.; data curation: I.Z. and H.F.; writing\u0026mdash;original draft: I.Z.; writing\u0026mdash;review and editing: all authors; visualization: I.Z. and N.B.; supervision: F.P. and P.A.; project administration: F.P. and P.A.; funding acquisition: J.D. and F.P. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNAseq data are accessible under the ArrayExpress accession E-MTAB-16008.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBron AJ, Paiva CS de, Chauhan SK, Bonini S, Gabison EE, Jain S, et al. Tfos dews II pathophysiology report. The ocular surface. 2017;15:438\u0026ndash;510.\u003c/li\u003e\n\u003cli\u003eSaito I, Terauchi K, Shimuta M, Nishiimura S, Yoshino K, Takeuchi T, et al. Expression of cell adhesion molecules in the salivary and lacrimal glands of Sjogren\u0026apos;s syndrome. Journal of clinical laboratory analysis. 1993;7:180\u0026ndash;7. doi:10.1002/jcla.1860070309.\u003c/li\u003e\n\u003cli\u003eWilliamson J, Gibson AA, Wilson T, Forrester JV, Whaley K, Dick WC. Histology of the lacrimal gland in keratoconjunctivitis sicca. 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Int J Mol Sci. 2020;21:1102. doi:10.3390/ijms21031102.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003ePrimer sequences.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"605\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e(q)PCR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eForward\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReverse\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmplicon\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003e18S\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eGGT GCA TGG CCG TTC TTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTGC CAG AGT CTC GTT CGT TA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e69 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eActa2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCAT CAC CAA CTG GGA CGA CA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eAGA GGC ATA GAG GGA CAG CA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e211 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eActb\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eGAT CCT CAC CGA GCG CGG CTA CA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eGCG GAT GTC CAC GTC ACA CTT CA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e298 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eAqp5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCAT GAA CCC AGC CCG ATC TT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCTC AGC GAG GAG GGG AAA AG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e144 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eAreg\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eAGA TAC ATC GAG AAC CTG GAG G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eAGA GAC AAA GAT AGT GAC AGC TAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e150 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eCreb5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCGA TAA CAG CCA TGC AGA AAG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCGA TGA GGA TGT AGT GAT GGT G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e128 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eCstb\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eGAT CTG TCT TCA GCT TCT CCG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTCG ATT CAA GCT GGG ACT TC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e134 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eEfna1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCAT CTC CAA ACC TAT CTA CCA TCA G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTGC AAA ACC TGT ACT TCC GG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e147 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eEgf\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eAGA AAC ACC AAG ACC CCA AG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTGT GCC CAT TCC ATC TAT GTG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e149 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eEpha2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eTGC CAG CGT CAG TAT TAA CC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTTC TTG CGG TAG GTG ACT TC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e141 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eFgf9\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eAAC TGG GCG GTT GGA ACA TA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTGT CAC AGA AGT GGG CTG AA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e119 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eFos\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTAC TAC CAT TCC CCA GCC GA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eGCT GTC ACC GTG GGG ATA AA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e113 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eFoxc1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eGGG CTT TCG TCT ATG ACT GTA G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTTC TGT TCG CTG GTG TGA G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e171 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eGdnf\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTGA AGA CCA CTC CCT CGG\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eGCT TGT TTA TCT GGT GAC CTT TTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e150 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eHbegf\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCCC CTA TAC ACA TAT GAC CAC AC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCAA CTT CAC TTT CTC TTC ACT TTC C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e150 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eJun\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTGG GCA CAT CAC CAC TAC AC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTCT GGC TAT GCA GTT CAG CC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e119 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eKit\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTGT GGC TAA AGA TGA ACC CTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eACA CTC CAG AAT CGT CAA CTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e135 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eKrt5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eAGA GGC TGA GTC CTG GTA C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eATG GCG TTC TGG AGG TTG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e177 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eLtf\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eATC TCT GTG CCC TGT GTA TTG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eGTT CCT AGC CCA CTC TTC AG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e191 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eLyz1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eGGA CTA GTG AGC TGT GCC TG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTTG CTC CTG TGG TTA TTG GCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e100 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eNgf\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCCC AAT AAA GGT TTT GCC AAG G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTTG CTA TCT GTG TAC GGT TCT G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e137 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003ePax6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCAG ATG CAA AAG TCC AGG TGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTTG GCT GCT AGT CTT TCC CG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e245 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003ePdgfra\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eGTT GCC TTA CGA CTC CAG ATG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTCA CAG CCA CCT TCA TTA CAG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e143 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eRab3d\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCTA TGC CGA TGA CTC CTT CAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eATG AGC AGG AAA CCC ATA GC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e180 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eSox10\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eAGC AGG CTG GAC ACT AAA CC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eAAA GGG GCA GCG ATG TGT TA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e147 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eVim\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTGA GAT CGC CAC CTA CAG GA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTTG CGC TCC TGA AAA ACT GC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e327 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e18S\u003c/em\u003e: 18S ribosomal RNA; \u003cem\u003eActa2\u003c/em\u003e: actin alpha 2 or alpha smooth muscle actin; \u003cem\u003eActb\u003c/em\u003e: \u0026beta;-actin; Aqp5: aquaporin 5; \u003cem\u003eAreg\u003c/em\u003e: amphiregulin; \u003cem\u003eCreb5\u003c/em\u003e: cyclic AMP-responsive element-binding protein 5; \u003cem\u003eCstb\u003c/em\u003e: cystatin B; \u003cem\u003eEfna1\u003c/em\u003e: ephrin A1; \u003cem\u003eEgf\u003c/em\u003e: epidermal growth factor; \u003cem\u003eEpha2\u003c/em\u003e: EPH receptor A2; \u003cem\u003eFgf9\u003c/em\u003e: fibroblast growth factor 9; \u003cem\u003eFos\u003c/em\u003e: Fos proto-oncogene; \u003cem\u003eFoxc1\u003c/em\u003e: Forkhead Box C1; \u003cem\u003eGdnf\u003c/em\u003e: Glial cell line-derived neurotrophic factor; \u003cem\u003eHbegf:\u003c/em\u003e heparin-binding EGF-like growth factor; \u003cem\u003eJun\u003c/em\u003e: Jun proto-oncogene; \u003cem\u003eKit\u003c/em\u003e: Kit proto-oncogene; \u003cem\u003eKrt5\u003c/em\u003e: keratin 5; \u003cem\u003eLtf\u003c/em\u003e: lactoferrin; \u003cem\u003eLyz1\u003c/em\u003e: lysozyme 1; \u003cem\u003eNgf\u003c/em\u003e: nerve growth factor: \u003cem\u003ePax6\u003c/em\u003e: paired box 6; \u003cem\u003ePdgfra\u003c/em\u003e: platelet derived growth factor receptor alpha; \u003cem\u003eRab3d\u003c/em\u003e: RAB3D, member RAS oncogene family; \u003cem\u003eSox10\u003c/em\u003e: SRY (sex determining region Y)-box 10; \u003cem\u003eVim\u003c/em\u003e: vimentin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eAntibodies.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"614\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibody\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConjugation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSolution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eManufacturer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eGoat Anti-Mouse IgG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eAlexa 488\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1:500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eA11029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003eThermo Fisher Scientific, Waltman, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eDonkey Anti-Rabbit IgG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eAlexa 488\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eA21206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003eThermo Fisher Scientific, Waltman, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u0026alpha;-SMA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eab124964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003eAbcam, Cambridge, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eAQP5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1:25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003esc-514022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology, Dallas, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eCYT5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1:50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003esc-32721\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology, Dallas, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eLTF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ePA5-95513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003eThermo Fisher Scientific, Waltman, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eFOXC1/2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1:100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eCPA3482\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003eCohesion Biosciences, London, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eVIM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1:200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1NP3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 255px;\"\u003e\n \u003cp\u003eCarl Roth, Karlsruhe, Germany\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026alpha;-SMA: or alpha smooth muscle actin; AQP5: aquaporin 5; CYT5: cytokeratin 5; LTF: lactoferrin; FOXC1/2: forkhead box protein C1/C2; VIM: vimentin\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"aqueous-deficient dry eye disease, dry eye disease, epithelial repair, growth factors, lacrimal gland regeneration, MAPK signaling","lastPublishedDoi":"10.21203/rs.3.rs-8077136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8077136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eAqueous-deficient dry eye disease (ADDE) results from lacrimal gland dysfunction leading to tear film instability and chronic ocular surface inflammation. Despite the gland\u0026rsquo;s known regenerative potential, the intrinsic molecular mechanisms enabling epithelial recovery remain poorly understood. This study aimed to characterize intrinsic regeneration processes of murine lacrimal gland epithelial cells (mLGECs) and identify pathways with therapeutic potential.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003ePrimary mLGECs were isolated from neonatal C57BL/6J mice and characterized by RT-PCR, immunocytochemistry, and β-hexosaminidase secretion assays. Acute injury was induced by brief exposure to 20% ethanol. Cell viability, apoptosis, and transcriptional responses were analyzed during early recovery phases. Pathway enrichment was conducted using Gene Ontology and KEGG annotations. Regeneration-associated growth factors were validated by quantitative PCR, ELISA, and functional assays assessing cell viability and wound closure in the presence or absence of the MAPK inhibitor SCH772984.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eCultured mLGECs preserved epithelial and lacrimal-specific markers and maintained regulated secretory activity. RNA-seq two hours post-injury revealed robust activation of MAPK signaling, accompanied by strong upregulation of regenerative ligands including Hbegf, Areg, Ngf, and Gdnf. Transient protein induction of HBEGF and AREG was confirmed. Functionally, HBEGF, AREG, NGF, and GDNF markedly enhanced epithelial survival, ATP recovery, and wound closure, while MAPK inhibition abolished regeneration, demonstrating pathway dependence.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese results identify MAPK-dependent mechanisms as central to the intrinsic regenerative response of lacrimal gland epithelial cells following acute injury. HBEGF and AREG emerged as key mediators driving epithelial survival and proliferation. The findings provide a mechanistic basis for developing defined growth factor\u0026ndash;based therapies as standardized, safe, and effective alternatives to blood-derived treatments for patients with severe ADDE.\u003c/p\u003e","manuscriptTitle":"Intrinsic Regeneration Mechanisms of Murine Lacrimal Gland Epithelial Cells for Therapeutic Applications in Aqueous- Deficient Dry Eye Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 14:07:27","doi":"10.21203/rs.3.rs-8077136/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65e3c180-6780-4082-b6fe-bbf66dcd1526","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T14:20:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 14:07:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8077136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8077136","identity":"rs-8077136","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}