Idiopathic pulmonary fibrosis (IPF) is a fatal, aging-related disease characterized by persistent lung fibroblast activation, progressive lung scarring, and several vascular abnormalities. We have previously demonstrated that aging-associated vascular dysfunction drives maladaptive endothelial responses to injury and exacerbates lung fibrosis via secretion of profibrotic endothelial cell-derived factors. However, regulatory mechanisms governing endothelial dysfunction during progressive lung fibrosis remain poorly understood. Here, using preclinical mouse models of progressive lung fibrosis as well as human IPF lungs, we demonstrate that miR-205-5p was overexpressed in lung endothelial cells (ECs) from fibrotic lungs and coordinated gene expression programs implicated in endothelial dysfunction and progressive fibrosis. Mechanistically, miR-205-5p induced senescence in lung ECs, mirroring the senescent phenotype of IPF lung ECs. Consistently, conditioned medium derived from lung ECs overexpressing miR-205-5p promoted lung fibroblast activation. Importantly, miR-205-5p inhibition in IPF lung ECs attenuated endothelial senescence and limited paracrine fibroblast activation. Finally, inhibition of miR-205-5p in vivo preserved the pulmonary vascular network and attenuated lung fibrosis progression in aged mice challenged with bleomycin. Collectively, our findings support what we believe to be a novel connection among lung endothelial miR-205-5p, endothelial senescence, and profibrotic alteration of the endothelial secretome and highlight miR-205-5p inhibition as a potential therapeutic intervention for pulmonary fibrosis.

The mammalian airway epithelium is a dynamic barrier essential for mucociliary clearance, gas exchange, and protection from environmental injury. Tight junction (TJ) proteins maintain epithelial integrity, yet their roles in coordinating repair and stem cell behavior are poorly defined. Claudin 18.1 (CLDN18.1), a lung-specific TJ protein highly expressed in alveolar epithelial cells and subsets of airway cells, has emerged as a regulator of epithelial homeostasis. Murine Cldn18 knockout models demonstrate an expansion of alveolar type II, club, and basal stem cell populations, implicating CLDN18.1 as a potential modulator of epithelial regeneration. Understanding how CLDN18.1 influences human airway epithelial differentiation and injury responses could be important for developing new strategies to enhance lung repair in chronic lung disease. Our preliminary data suggests lower expression in cystic fibrosis donor bronchial regions compared to healthy controls. Based on this observation we hypothesized that loss of CLDN18.1 promotes goblet cell differentiation by altering club-cell fate decisions, particularly under inflammatory conditions. To test this, we generated CRISPR–Cas9–mediated CLDN18.1/18.2 knockout (KO) human primary bronchial epithelial cells (HBECs) using gRNAs targeting exon 2. Knockout efficiency was confirmed by Sanger sequencing, qPCR, and Western blot. HBECs were differentiated at air–liquid interface for 28 days, and epithelial composition was quantified by confocal microscopy and image quantification and qRT-PCR. Data shows that CLDN18.1 loss in HBEC isolated from donors with no prior evidence of lung disease does not significantly alter baseline differentiation into ciliated, club, or mucus cell lineages, contrary to expectations based on prior studies. Quantification across multiple fields revealed comparable proportions of epithelial cell types in KO and GFP controls; however, CLDN18.1-deficient cultures exhibited a significant increase in total cell number. This proliferative phenotype is consistent with CLDN18.1’s known tumor-suppressive function and supports the effectiveness of the knockout. In conclusion our data suggest that downregulation of CLDN18 supports a proliferative response important for normal epithelial repair allowing BCs to exit a barrier maintaining state and chronic CLDN18 downregulation that we have observed in end-stage disease may be due to an ongoing perception of injury in the cellular microenvironment. Ongoing experiments will clarify the temporal-spatial regulation of CLDN18.1 and how it integrates inflammatory cues to regulate proliferation and epithelial fate. Ultimately, defining CLDN18.1’s function in human airway biology may enable targeted approaches to enhance epithelial regeneration in chronic lung disease. This work was funded by an American Lung Foundation Grant, awarded to ALR This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Idiopathic pulmonary fibrosis (IPF) is a lethal progressive lung disease urgently needing new therapies. Current treatments only delay disease progression, leaving lung transplant as the sole remaining option. Recent studies support a model whereby IPF arises because alveolar epithelial type II (AT2) cells, which normally mediate distal lung regeneration, acquire airway and/or mesenchymal characteristics, preventing proper repair. Mechanisms driving this abnormal differentiation remain unclear. We performed integrated transcriptomic and epigenomic analysis of purified AT2 cells which revealed genome-wide alterations in IPF lungs. The most prominent epigenetic alteration was activation of an enhancer in thyroid receptor interactor 13 (TRIP13), although TRIP13 was not the most significantly transcriptionally upregulated gene. TRIP13 is broadly implicated in epithelial-mesenchymal plasticity. In cultured human AT2 cells and lung slices, small molecule TRIP13 inhibitor DCZ0415 prevented acquisition of the mesenchymal gene signature characteristic of IPF, suggesting TRIP13 inhibition as a potential therapeutic approach to fibrotic disease.

Introduction: The landscape of electronic (e)-cigarettes has rapidly evolved and many flavored 4th generation devices are available. E-liquids contain humectants, propylene glycol and glycerin, flavorant chemicals, and nicotine. While flavoring chemicals are generally recognized as safe for gastrointestinal consumption, flavors have been shown to have detrimental effect on cellular functions following inhalation. We previously conducted bulk RNAseq on mouse lungs across different types of e-cigarette exposures. However, transcriptional changes to specific cell types are difficult to assess using bulk RNAseq. Deconvolution is a computational method that uses a reference gene matrix to estimate the relative proportions of different cell types in a bulk sample. We applied this method to our bulk RNAseq data to obtain further insight into cellular responses to e-cigarettes. Methods: Female C57BL/6 mice (8-weeks-old) were exposed to tobacco, menthol, mint, or mango-flavored JUUL aerosols, or room air for 1 hour/d, 5 d/week, x4 weeks using the InExpose System (Scireq, Emka). Lung RNA was isolated for bulk RNAseq. Differential gene expression (DGE) analysis was performed using DESeq2. Ingenuity Pathway Analysis was performed on significant differentially expressed genes (DEGs). A reference single-cell RNAseq dataset from postnatal day 28 murine lungs was used to construct a gene signature matrix for deconvolution of bulk RNAseq data in CIBERSORTx using default settings. Results: Tobacco and menthol e-cigarette exposures had the greatest number of DEGs. Out of the different flavors, 3 out of the 4 groups (menthol, tobacco, and mango) showed overlap in transcriptional changes such as downregulation of the alpha and beta globin chain genes (HBA1/HBA2 and HBB) and upregulation of Neutral cholesterol ester hydrolase 1 (Nceh1) gene. Mint exposure was associated with 1 DEG, Heat shock protein 1B (Hspa1b). Based on the deconvolution, the inferred proportion of alveolar type 2 cells and arterial endothelial cells were decreased in the tobacco and menthol groups compared to air controls. Conversely, the proportion of alveolar type 1 cells and B cells were estimated to be higher in the tobacco and menthol groups compared to air controls. Conclusion: Daily tobacco and menthol flavored e-cigarette exposures were associated with many transcriptomic changes within the lungs. These transcriptomic changes translated into inferred alterations in cell proportions of alveolar type 1 and 2 cells, arterial endothelial cells, and B cells. These data suggest that these cell types are either exhibiting an altered transcriptional state or have changed in proportions. Further studies are needed for validation of these findings.

Rationale : Tight junction (TJ) protein claudin-18 (CLDN18) is extensively studied in the alveolar epithelium, but its role in the airway has not yet been elucidated. Interestingly, reduction of CLDN18 has been linked to the development of asthma and CLDN18 is downregulated in human primary small airway epithelial cells exposed to cigarette smoke, suggesting possible involvement in human airway disease. Chronic exposure to environmental irritants, e.g., cigarette smoke, is a major risk factor for chronic obstructive pulmonary disease (COPD), causing airway remodeling and TJ disruption. Here we investigated expression and function of CLDN18 in mouse distal airway homeostasis and regeneration. Methods: Wild type (WT) and global Cldn18 KO mice were injected intraperitoneally with naphthalene (NAP) (225 mg/kg) to model airway injury. Lungs were analyzed at day 0, 3 (maximal injury), 7 (beginning of regeneration), and 21 (full regeneration) post-NAP by Hematoxylin and eosin, Periodic Acid-Schiff and immunofluorescence (IF) staining. Single cell RNA-sequencing (scRNA-seq) was performed at days 0, 3 and 14 post-NAP. Three-dimensional (3D) spheroids were derived from sorted airway cells (CD45-/CD31-/EpCAM+/CD24+). Results: In uninjured WT mice, CLDN18 was expressed in a subset of distal airway ciliated cells, by both IF and scRNA-seq, and rarely in club cells. Absence of CLDN18 significantly altered the cellular composition of the airways: mice lacking CLDN18 (Cldn18 KO) exhibited a dramatic increase in pulmonary neuroendocrine cells (PNECs) and neuroendocrine bodies (NEBs). In injured WT mice, CLDN18 became transiently widespread at day 3 (D3) post-NAP in all the ciliated cells covering the denuded epithelium. Unexpectedly, Cldn18 KO mice exhibited goblet cell metaplasia (GCM) at D21 after NAP when WT mice showed full regeneration. Airway 3D spheroids from Cldn18 KO mice also showed an increase in goblet cells compared to WT. ScRNA-seq analysis revealed that expression of Cldn18 and a ligand of Notch, Jag1, is mutually exclusive in the WT ciliated population at baseline. Following injury, Cldn18 KO ciliated cells overexpress Jag1 and Notch1, suggesting that CLDN18 might restrict GCM via inhibiting Notch signaling. Conclusions: Our findings reveal a novel function for CLDN18 and suggest a key role for ciliated cells in airway regeneration Furthermore, a novel CLDN18-Notch interaction which may be either cell autonomous or non-cell autonomous, may be critical to restrict goblet cell differentiation in both normal and regenerating airways. Since COPD is characterized by GCM, understanding mechanisms of GCM onset downstream of CLDN18 has relevance for the identification of novel therapies for COPD.

ABSTRACTLung adenocarcinoma (LUAD) is the most common subtype of cancer arising in the distal lung. LUAD encompasses several pathologic subtypes, each with differing clinical outcomes and biological behaviors. However, the molecular and cellular underpinnings of the different subtypes are largely unknown. Understanding which cell populations in the distal lung contribute to LUAD could provide insights into the marked heterogeneity in pathologic features, clinical presentation and responses to therapy of LUAD. Differential expression analysis of lung adenocarcinoma transcriptomes from The Cancer Genome Atlas revealed distinct alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cell signatures within human LUAD with significantly different survival outcomes between tumors expressing AT2 and AT1 gene signatures, suggesting AT1 cells might contribute to a subset of LUAD cases. To address this, we tested the ability of AT1 cells to give rise to LUAD following induction of KrasG12D, a known oncogenic driver of human LUAD. Activation of KrasG12Din Gram-domain containing 2 (Gramd2)+AT1 cells gave rise to multiple LUAD lesions, primarily of papillary histology. In contrast, activation of KrasG12Din surfactant protein C (Sftpc+) AT2 cells resulted in LUAD lesions of lepidic histology. Immunohistochemistry established thatGramd2:KrasG12Dlesions were of primary lung origin and not metastatic events. Spatial transcriptomic profiling revealed distinct pathway alterations within Gramd2- and Sftpc-derived LUAD. Immunofluorescence confirmed differences observed in the spatial transcriptomic analysis in expression patterns and distribution of cell-specific markers depending on cell of origin, while universal upregulation of the Krt8 intermediate cell state marker was observed. Our results are consistent with Gramd2+AT1 cells serving as a putative cell of origin for LUAD and suggest that LUAD may be a collection of adenocarcinomas that share a common location within the distal lung but arise from different cells of origin.

Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and presents clinically with a high degree of biological heterogeneity and distinct clinical outcomes. The current paradigm of LUAD etiology posits alveolar epithelial type II (AT2) cells as the primary cell of origin, while the role of AT1 cells in LUAD oncogenesis remains unknown. Here, we examine oncogenic transformation in mouse Gram-domain containing 2 (Gramd2)+ AT1 cells via oncogenic KRASG12D. Activation of KRASG12D in AT1 cells induces multifocal LUAD, primarily of papillary histology. Furthermore, KRT8+ intermediate cell states were observed in both AT2- and AT1-derived LUAD, but SCGB3A2+, another intermediate cell marker, was primarily associated with AT1 cells, suggesting different mechanisms of tumor evolution. Collectively, our study reveals that Gramd2+ AT1 cells can serve as a cell of origin for LUAD and suggests that distinct subtypes of LUAD based on cell of origin be considered in the development of therapeutics.

Alveolar epithelial regeneration is critical for normal lung function and becomes dysregulated in disease. While alveolar type 2 (AT2) and club cells are known distal lung epithelial progenitors, determining if alveolar epithelial type 1 (AT1) cells also contribute to alveolar regeneration has been hampered by lack of highly specific mouse models labeling AT1 cells. To address this, the Gramd2 CreERT2 transgenic strain was generated and crossed to Rosa mTmG mice. Extensive cellular characterization, including distal lung immunofluorescence and cytospin staining, confirmed that GRAMD2 + AT1 cells are highly enriched for green fluorescent protein (GFP). Interestingly, Gramd2 CreERT2 GFP + cells were able to form organoids in organoid co-culture with Mlg fibroblasts. Temporal scRNAseq revealed that Gramd2+ AT1 cells transition through numerous intermediate lung epithelial cell states including basal, secretory and AT2 cell in organoids while acquiring proliferative capacity. Our results indicate that Gramd2+ AT1 cells are highly plastic suggesting they may contribute to alveolar regeneration.

SUMMARY Expansion of pulmonary neuroendocrine cells (PNECs) is a pathological feature of many human lung diseases. Human PNECs are inherently difficult to study due to their rarity (<1% of total lung cells) and a lack of established protocols for their isolation. We used induced pluripotent stem cells (iPSCs) to generate induced PNECs (iPNECs), which express core PNEC markers, including ROBO receptors, and secrete major neuropeptides, recapitulating known functions of primary PNECs. Furthermore, we demonstrate that differentiation efficiency is increased in the presence of an air-liquid interface and inhibition of Notch signaling. Single-cell RNA sequencing (scRNA-seq) revealed a PNEC-associated gene expression profile that is concordant between iPNECs and human fetal PNECs. In addition, pseudotime analysis of scRNA-seq results suggests a basal cell origin of human iPNECs. In conclusion, our model has the potential to provide an unlimited source of human iPNECs to explore PNEC pathophysiology associated with several lung diseases.

Abstract Lung adenocarcinoma (LUAD) is the most common subtype of cancer arising in the distal lung. LUAD encompasses several pathologic histologies, some with important differing clinical outcomes and biological behaviors. However, the molecular and cellular underpinnings of the different subtypes are largely unknown. Understanding which cell populations in the distal lung contribute to LUAD could provide insights into the marked heterogeneity in pathologic features, clinical presentation and responses to therapy of LUAD. Differential expression analysis of LUAD transcriptomes from The Cancer Genome Atlas revealed distinct alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cell signatures with significantly different survival outcomes between tumors expressing AT2 and AT1 gene signatures. The data suggests that AT1 cells might contribute to a subset of LUAD cases. To determine if AT1 cells could give rise to LUAD, we utilized transgenic mouse models to induce KrasG12D, a known oncogenic driver of human LUAD, in Gram-domain containing 2 (Gramd2)+ expressing AT1 cells. This gave rise to multiple LUAD lesions, as confirmed by micro computed tomography and pathologist-evaluated hematoxylin and eosin staining, primarily of papillary histology. In contrast, activation of KrasG12D in surfactant protein C (Sftpc+) AT2 cells resulted in LUAD lesions of exclusively lepidic histology. Immunohistochemistry established that Gramd2:KrasG12D lesions were of primary lung origin and not metastatic events. Spatial transcriptomic profiling revealed distinct pathway alterations within Gramd2- and Sftpc-derived LUAD, including specific upregulation of TGFβ-mediated epithelial to mesenchymal transition (EMT) in Gramd2+ AT1 LUAD. Immunofluorescence confirmed differences observed in the spatial transcriptomic analysis in expression patterns and distribution of cell-specific markers depending on cell of origin, while universal upregulation of a Krt8+ intermediate cell state marker was observed. Our results are consistent with Gramd2+ AT1 cells serving as a putative cell of origin for LUAD and suggest that LUAD may be a collection of adenocarcinomas that share a common location within the distal lung but arise from different cells of origin, a finding with potentially important therapeutic implications. Citation Format: Minxiao Yang, Hua Shen, Per Flodby, Michael Koss, Rania Bassiouni, Yixin Liu, Theresa Ryan Stueve, Daniel J. Mullen, Amy L. Ryan, Tea Jashashvili, John Carpten, Alessandra Castaldi, W. Dean Wallace, Beiyun Zhou, Zea Borok, Crystal N. Marconett. Alveolar epithelial type 1 cells can serve as a cell of origin for lung adenocarcinoma with distinct molecular and phenotypic presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5.

Alveolar epithelial type I (AT1) cells are large, squamous cells with elongated thin cytoplasmic processes covering the majority of the gas exchange surface of the lung that play an active role in alveolar homeostasis. AT1 cells are derived mainly from AT2 cells during development and following injury, but also arise from other progenitors. AT1 cells constitute a heterogeneous population and while initially considered to be terminally differentiated, in certain circumstances, they can both self-renew and differentiate into AT2 cells. Next generation sequencing and lineage tracing studies have set the stage for more in-depth characterization of AT1 cell function and biology.

BACKGROUND:Selective proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during eukaryotic development, generating a cleaved histone H3 (H3cl) product within a small, but significant, portion of the genome. Although increasing evidence supports a regulatory role for H3NT proteolysis in gene activation, the nuclear H3NT proteases and the biological significance of H3NT proteolysis remain largely unknown.RESULTS:In this study, established cell models of skeletal myogenesis were leveraged to investigate H3NT proteolysis. These cells displayed a rapid and progressive accumulation of a single H3cl product within chromatin during myoblast differentiation. Using conventional approaches, we discovered that the canonical extracellular matrix (ECM) protease, matrix metalloproteinase 2 (MMP-2), is the principal H3NT protease of myoblast differentiation that cleaves H3 between K18-Q19. Gelatin zymography demonstrated progressive increases in nuclear MMP-2 activity, concomitant with H3cl accumulation, during myoblast differentiation. RNAi-mediated depletion of MMP-2 impaired H3NT proteolysis and resulted in defective myogenic gene activation and myoblast differentiation. Supplementation of MMP-2 ECM activity in MMP-2-depleted cells was insufficient to rescue defective H3NT proteolysis and myogenic gene activation.CONCLUSIONS:This study revealed that MMP-2 is a novel H3NT protease and the principal H3NT protease of myoblast differentiation. The results indicate that myogenic signaling induces MMP-2-dependent H3NT proteolysis at early stages of myoblast differentiation. Importantly, the results support the necessity of nuclear MMP-2 H3NT protease activity, independent of MMP-2 activity in the ECM, for myogenic gene activation and proficient myoblast differentiation.