Citations

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 Rationale Systemic sclerosis (SSc) is a rare, chronic autoimmune disease characterized by vasculopathy and fibrosis of the skin and internal organs. Among its manifestations, interstitial lung disease (ILD) is the leading cause of mortality, yet its clinical course is variable, ranging from stable to progressive pulmonary fibrosis (PPF). The 2022 ATS/ERS/JRS/ALAT guidelines defined progressive PPF as a phenotype of non- Idiopathic Pulmonary Fibrosis (IPF) - ILDs that behave similarly to IPF. Although a subset of SSc-ILD patients develop PPF, biomarkers predicting progression remain lacking. We and others have shown that endothelial dysfunction contributes to the progression of fibrotic lung disease, and that the pulmonary vasculature, continuously exposed to the bloodstream, is influenced by soluble circulating factors, including circulating extracellular vesicles (cEVs), which carry bioactive cargo capable of modulating endothelial behavior. Here, we investigated whether cEVs from SSc patients with distinct fibrotic phenotypes differentially influence human lung endothelial cells (ECs), promoting a pro-fibrotic endothelial phenotype. Methods plasma samples were collected from five groups: 1) Healthy controls (n = 4); 2) SSc-noILD (no high-resolution-CT (HCRT( or functional evidence of ILD at time of enrollment, n = 4); 3) SSc-ILD (HCRT and functional evidence of ILD without meeting the PPF criteria at time of sampling, n = 3); 4) SSc-PPF (meeting the PPF criteria, plasma collected prior to anti-fibrotic therapy, n = 4); 5) IPF (plasma collected prior to anti-fibrotic therapy, n = 3). cEVs were isolated using size-exclusion-chromatography and characterized using Nanoparticle Tracking Analysis (NTA). Human lung ECs were treated with cEVs from the five groups (2x109 particles/ml, 48 hours), followed by qPCR for markers selected for their established involvement in endothelial dysfunction during lung fibrotic progression. Results NTA showed no significant differences in plasma cEV concentrations across the five groups. Human lung ECs exposed to cEVs from SSc or SSc-ILD patients exhibited no significant gene expression changes compared to healthy controls. In contrast, cEVs from SSc-PPF patients markedly downregulated endothelial identity (PECAM1, ERG, TEK), barrier integrity (CDH13, S1PR1), and basement membrane (COL4A1, HSPG2) genes, similarly to changes induced by cEVs from IPF patients. Conclusion cEVs from SSc-PPF patients induce pro-fibrotic transcriptional changes in lung ECs resembling those induced by IPF cEVs and distinct from SSc-ILD cEVs, suggesting that EVs carry cargo that both reflect and influence fibrosis progression. These findings highlight the potential of cEVs as biomarkers for identifying SSC-ILD patients at risk of progression and highlight the clinical relevance of future studies aimed at characterizing their cargo. This abstract is funded by: James Depauw Grant for Pilot Projects

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.

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 RATIONALE: Vascular dysfunction contributes to pathological tissue remodeling in aging-associated disease such as Idiopathic Pulmonary Fibrosis (IPF). IPF is an incurable lung disease characterized by progressive accumulation of scar-forming fibroblasts and several vascular alterations, including endothelial transcriptional changes leading to a pro-fibrotic endothelial secretory phenotype. To date, the mechanisms governing these transcriptional changes remain largely unexplored. To fill this gap, we sought to investigate whether microRNAs (miRNAs), small non-coding RNA that function as key gene expression regulators, modulate the transcriptional changes of the endothelium associated with progressive pulmonary fibrosis. METHODS AND RESULTS: miRNA expression profiling of lung ECs (LECs) sorted from aged mice with persistent lung fibrosis revealed upregulation of miR-205-5p. Further analysis demonstrated that endothelial miR-205-5p was strongly upregulated in an additional mouse model of progressive lung fibrosis induced by repetitive bleomycin dose as well as in human LECs purified from IPF lungs. RNA-seq analysis of normal human LECs overexpressing miR-205-5p revealed reduction of genes associated to endothelial identity (e.g. PECAM1, ERG, NOS3), and an increase of genes encoding for soluble pro-inflammatory and pro-fibrotic molecules, including TGFBI, TGFB2, TGFB3, CXCL10, CCL5, IL1B and FAS compared to normal LECs transfected with a control mimic. Consistently, conditioned medium from miR-205-5p overxpressing LECs exerted a pro-fibrotic activity on quiescent human lung fibroblasts, as demonstrated by upregulation of a panel of profibrotic transcripts (e.g. COL1A1, CTHRC1, FN1 and TNC) and elevated Collagen I deposition compared to human lung fibroblasts exposed to conditioned medium generated by human LECs transfected with a control mimic. CONCLUSION: Altogether, our findings suggest that lung endothelail miR-205-5p induces loss of endothelial cell identity and drives the secretion of pro-fibrotic molecules that promote the transition of quiescent fibroblasts into scar-forming fibroblast through paracrine signal. Our data set the stage for further investigations into the role of miR-205-5p and associated pathways in lung vascular injury responses, with the long term goal of targeting endothelial dysfunction for slowing the progression of 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.