Gilbert, Rachel M.

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.