Tschumperlin, Daniel J.

Abstract Lung fibrosis, including idiopathic pulmonary fibrosis (IPF), represents a spectrum of progressive interstitial lung diseases characterized by disrupted epithelial repair, fibroblast activation, and excess extracellular matrix accumulation. A central feature of fibrotic progression is the loss of alveolar type 2 epithelial (AT2) cell identity and the emergence of aberrant transitional states that fail to support normal regeneration. Based on our re-analysis of multiple publicly available scRNA-seq datasets from the IPF Cell Atlas, S100A2 expression is tightly associated with basal-like reprogramming of AT2 cells and is a top upregulated gene at both mRNA and protein levels in IPF lungs. Furthermore, scRNA-seq data from human lung organoid models reveal that AT2 cells co-cultured with fibroblasts acquire a basal-like phenotype and express high levels of S100A2. Functionally, overexpression of S100A2 in human iPSC-derived lung alveolar epithelial type 2 (iAT2) cells leads to loss of AT2 cellular identity, increased generation of reactive oxygen species, and activation of RAGE signaling pathway. We demonstrate that pharmacologic inhibition of RAGE using Azeliragon preserves AT2 cell populations, associated with reduced oxidative stress in iAT2 cells, and significantly attenuates collagen deposition in bleomycin-induced lung fibrosis models. Collectively, our results indicate that S100A2 is a key driver of epithelial dysfunction in lung fibrosis, promoting loss of AT2 identity, aberrant basal fate acquisition, and persistent epithelial injury. Targeting the unrecognized S100A2–RAGE pathway may offer a new therapeutic strategy to restore epithelial homeostasis in lung fibrosis.

Abstract The lung, one the largest branching organs in our body and vital for survival at first breath, is commonly known as the gas-exchange organ. Recent novel findings on lung sensing cells, molecules, and lung-brain crosstalk in the emerging discipline of interoception have highlighted the consideration of the lung as a sensory organ. Acknowledging the central importance of lung sensing to human health and disease, NHLBI convened a workshop to synthesize the past, current, and future of lung sensing research, with in-person presentations by ∼20 interdisciplinary experts and a large virtual audience. Here, we highlight the key topics discussed and summarize a blueprint for the future in this important field.

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.

Abstract Human genomic studies link reduced CUB domain-containing protein 1 (CDCP1) expression with myocardial recovery in heart failure. While CDCP1 regulates cardiac fibroblast proliferation in vitro, it’s in vivo role in cardiac fibrosis remains unclear. Using a Cdcp1 -knockout (KO) angiotensin II/phenylephrine mouse model, we show that Cdcp1 deletion reduces echocardiographic left ventricular mass, histologic cardiac fibrosis, and pro-fibrotic gene expression, along with decreased fibroblast activation and inflammatory markers. Spatial transcriptomics identified a pressure overload–expanded fibroblast subpopulation enriched for growth factor and TGF-β signaling (FB5), which was markedly attenuated in Cdcp1 -KO hearts, alongside reduction of a pro-inflammatory cardiomyocyte subtype (CM4). Complementary studies in human ventricular fibroblasts demonstrate that CDCP1 knockdown reduced extracellular matrix gene expression and collagen I deposition. These findings establish CDCP1 as a regulator of cardiac fibrotic remodeling in vivo and open avenues for its further investigation as a potential therapeutic target.

Abstract Lung fibroblasts are key regulators of tissue homeostasis and extracellular matrix (ECM) remodeling, and their aberrant activation drives the progressive parenchymal scarring characteristic of idiopathic pulmonary fibrosis (IPF), a fatal disease with limited therapeutic options. Despite their central pathogenic role, lung fibroblasts are difficult to isolate due to their embedded position within the ECM, and standard in vitro culture conditions may lead to the loss of their native functional and transcriptional characteristics, hampering the study of fibroblast behavior in disease. The transcriptional heterogeneity of lung fibroblast subtypes and the extent to which culture-induced alterations diverge from native tissue signatures remain poorly understood. Here, we integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics of lung tissue from IPF patients and age-matched healthy donors with transcriptomic profiling of cultured fibroblasts collected at passages 1 and 6 after isolation using three optimized protocols: whole lung cell suspension (WLCS), negative fraction enrichment, and outgrowth. Tissue-based analysis identified six transcriptionally distinct mesenchymal subtypes: alveolar, adventitial, inflammatory, peribronchial, CTHRC1+ and smooth muscle cell (SMC). The fibroblast subtype CTHRC1+ represented the most transcriptionally activated pro-fibrotic subtype, showing the greatest upregulation of ECM biosynthesis genes, a prominent role in intercellular communication, and preferential enrichment within fibroblastic foci in IPF lung tissue. Pseudotime trajectory analysis supported a directional transcriptional continuum from alveolar and inflammatory fibroblasts toward the CTHRC1+ state, driven by coordinated activation of pro-fibrotic transcription factors, including RUNX2, CREB3L1, and SCX. In vitro culture progressively reshaped fibroblast transcriptional identity relative to native tissue, with increased collagen and matrix metalloproteinase (MMP) expression during passaging, loss of distinct CTHRC1+ fibroblasts, and gain of alveolar fibroblasts displaying pro-fibrotic activation across all isolation protocols. These findings provide a high-resolution transcriptional map of lung fibroblast heterogeneity in IPF and highlight critical limitations of standard in vitro culture systems for recapitulating native fibroblast diversity, with important implications for the development and evaluation of fibroblast-targeted therapeutic strategies in IPF.

Abstract Pulmonary fibrosis (PF) involves excessive collagen accumulation, yet mechanisms shifting the balance of synthesis and degradation toward net deposition remain unclear. Myeloperoxidase (MPO) inversely correlates with survival in PF. Using the bleomycin model, we found MPO knockout (MPOko) mice were protected from fibrosis, and pharmacological MPO inhibition after peak inflammation (day 7) recapitulated this protection. MPO persisted in lung tissue 21 days post-injury despite neutrophil efflux, linking acute inflammation to sustained remodeling. Mechanistically, we identified that MPO inhibits Cathepsin K (CatK), a potent collagenolytic enzyme involved in fibrosis resolution. Notably, CatK gene expression ( CTSK ) is elevated in PF, suggesting post-translational inhibition of CatK. MPOko and inhibitor-treated mice exhibited elevated CatK activity after bleomycin; exogenous addition of pathophysiologic concentrations of MPO reduced CatK activity in mouse precision-cut lung slices and human fibroblasts. Biochemically, MPO reduced CatK activity to 33% of control. In two distinct cohorts of PF patients, we observed significantly increased MPO protein levels in platelet poor plasma and in lung tissue. In PF patients, 62% had MPO levels in platelet poor plasma exceeding healthy controls, while lung tissue from other PF patients showed significantly elevated MPO staining. Plasma levels were inversely correlated with decreased survival, FVC, and DLCO. These findings establish MPO as a post-translational inhibitor of CatK-mediated collagenolysis, revealing a mechanism linking acute inflammation to sustained fibrosis and suggest a patient subpopulation that may benefit from MPO-targeted therapy. Graphical Abstract Myeloperoxidase persists in lung tissue after injury and inhibits cathepsin K activity, impairing collagen degradation and promoting extracellular matrix accumulation during pulmonary fibrosis.

Abstract Repetitive injury is hypothesized to lead to progressive tissue fibrosis and end-stage organ failure. Whether tissue-resident mesenchymal cell populations retain epigenetic memory of prior injuries that contribute to this pathological process is unknown. Here we used a genetic lineage labeling approach to mark the lung mesenchyme prior to injury, then performed multimodal analyses on isolated lung mesenchyme during the initiation, progression, and resolution of the fibrotic response. Our results demonstrate the remarkable epigenetic and transcriptional plasticity of the lung mesenchyme during fibrotic activation and de-activation. Despite this plasticity, we also find that the lung mesenchyme exhibits an enhanced fibrotic program upon reinjury. We identify RUNX1 as a critical driver of both fibrotic activation and fibrotic memory. Comparison of fresh isolated and cultured lung mesenchyme demonstrates that RUNX1 is spontaneously activated in standard culture conditions, previously masking these roles of RUNX1. Targeted knockdown of RUNX1 dampens fibrotic mesenchymal cell activation immediately after cell isolation, but with reduced efficacy after only days of culture, confirming its functional importance to both early activation and long-term memory. Collectively, our findings implicate RUNX1 in the initiation and memory of fibrotic mesenchymal cell activation that together prime enhanced mesenchymal cell responses upon repeated injury.

This study uncovers a previously unrecognized role for NAV3 in TGF-β1-driven cardiac fibroblast activation. We show that NAV3 facilitates profibrotic remodeling through noncanonical signaling and cell cycle arrest, independently of SMAD2/3. These findings position NAV3 as a novel regulator of fibroblast phenotype and a potential modulator of cardiac fibrosis.

Pulmonary fibrosis is a devastating and progressive disease marked by replacement of gas-exchanging tissue with collagen-rich scar. The mechanical environment is profoundly altered in pulmonary fibrosis and contributes to disease progression via feedback relationships between cells, the extracellular matrix, and the evolving mechanical environment. Targeting these mechanobiological feedback loops has emerged as a promising approach to interrupt disease progression, though with challenges in how to intervene selectively, safely, and effectively. We posit that further delineation of cell–matrix mechanobiological interactions will be pivotal to promoting fibrosis resolution and should guide efforts to discover and implement new approaches that can preserve or even restore lung function. To set the stage for these advances, we first review the mechanobiology of the healthy lung and the feedback loops that promote fibrosis progression. We then lay out the challenges and opportunities for targeting the fibrotic matrix as an essential element for protecting or restoring lung function.

Recent studies highlight transcriptionally distinct fibroblast subpopulations in human lungs. We observed the loss of these native transcriptional programs as fresh isolated cells are maintained in traditional culture conditions. Identifying the signals defining native fibroblast identities will be pivotal to creating culture models that preserve unique subpopulations. The screening system developed here will allow the investigation of a broad selection of cues, leading to better culture models for studying human lung fibroblast function and plasticity.