Link, Patrick A

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.

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.

Abstract Pulmonary fibrosis is increasingly understood to involve dysfunction within and across multiple cellular compartments, with recent attention highlighting the involvement of pulmonary vascular dysfunction in failed repair and progression of fibrosis. Formulation and delivery of lung-targeting lipid nanoparticles (LNPs) may provide a means to selectively target the lung but not systemic vasculature. However, the feasibility and efficacy of such approaches in the fibrotic lung are unknown. We sought to test whether intravenously administered lung-targeting LNPs can safely deliver mRNA to the healthy and fibrotic lung vasculature in young and aged mice and whether delivery of mRNA encoding a matricellular protein could promote fibrosis resolution. We used a selective organ targeting LNP formulation and characterized cell-specificity of delivery after bleomycin-induced lung fibrosis. We then delivered Ccn3 mRNA (encoding cellular communication network factor 3) to aged mice in the setting of established lung fibrosis and evaluated fibrotic regression and vascular repair. The matricellular protein encoded by Ccn3 was previously identified by our group as an important regulator of lung endothelial function. We found that LNP delivery was lung specific and predominantly endothelial targeting in the setting of lung fibrosis. Delivery of Ccn3 mRNA to aged mice via LNPs modestly reduced fibrosis and improved microvascular density in the lungs. Our results support the concept that cell-specific and repair-promoting cargos delivered via lung-targeting LNPs may have utility for treatment of established fibrosis.

This study demonstrates that circulating extracellular vesicles (cEVs) isolated from young mice reverse the transcriptional alterations of the aged mouse pulmonary vasculature, leading to a more youthful endothelial transcriptional phenotype. As a result of this vascular phenotype, aged mice are protected from bleomycin-induced pulmonary fibrosis. These findings highlight the therapeutic potential of targeting vascular aging to alleviate pulmonary fibrosis.

Abstract Idiopathic pulmonary fibrosis (IPF) is an aggressive and, thus far, incurable disease characterized by aberrant fibroblast-mediated extracellular matrix deposition. Our understanding of the disease etiology is incomplete; however, there is consensus that a reduction-oxidation (redox) imbalance plays a role. In this study, we use the autofluorescent properties of two redox molecules, NAD(P)H and FAD, to quantify changes in their relative abundance in living lung tissue of mice with experimental lung fibrosis and in freshly isolated cells from mouse lungs and humans with IPF. Our results identify cell population–specific intracellular redox changes in the lungs in experimental and human fibrosis. We focus particularly on redox changes within collagen-producing cells, where we identified a bimodal distribution of NAD(P)H concentrations, establishing NAD(P)Hhigh and NAD(P)Hlow subpopulations. NAD(P)Hhigh fibroblasts exhibited elevated profibrotic gene expression and decreased collagenolytic protease activity relative to NAD(P)Hlow fibroblasts. The NAD(P)Hhigh population was present in healthy lungs but expanded with time after bleomycin injury, suggesting a potential role in fibrosis progression. We identified a similar increased abundance of NAD(P)Hhigh cells in freshly dissociated lungs of subjects with IPF relative to control subjects, as well as similar reductions in collagenolytic activity in this cell population. These data highlight the complexity of redox state changes in experimental and human pulmonary fibrosis and the need for selective approaches to restore redox imbalances in the fibrotic lung.

AbstractPulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell‐specific approach. Our previous work demonstrated that targeting non‐canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non‐canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH‐associated PASMC remodeling, with longer‐lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non‐canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH.

Aberrant vascular remodeling contributes to the progression of many aging-associated diseases, including idiopathic pulmonary fibrosis (IPF), where heterogeneous capillary density, endothelial transcriptional alterations, and increased vascular permeability correlate with poor disease outcomes. Thus, identifying disease-driving mechanisms in the pulmonary vasculature may be a promising strategy to limit IPF progression. Here, we identified Ccn3 as an endothelial-derived factor that is upregulated in resolving but not in persistent lung fibrosis in mice, and whose function is critical for vascular homeostasis and repair. Loss and gain of function experiments were carried out to test the role of CCN3 in lung microvascular endothelial function in vitro through RNAi and the addition of recombinant human CCN3 protein, respectively. Endothelial migration, permeability, proliferation, and in vitro angiogenesis were tested in cultured human lung microvascular endothelial cells (ECs). Loss of CCN3 in lung ECs resulted in transcriptional alterations along with impaired wound-healing responses, in vitro angiogenesis, barrier integrity as well as an increased profibrotic activity through paracrine signals, whereas the addition of recombinant CCN3 augmented endothelial function. Altogether, our results demonstrate that the matricellular protein CCN3 plays an important role in lung endothelial function and could serve as a promising therapeutic target to facilitate vascular repair and promote lung fibrosis resolution.