Raslan, Ahmed

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: 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 characterized by the sustained activation of interstitial fibroblasts leading to excessive collagen deposition and progressive organ failure. Epigenetic and metabolic abnormalities have been shown to contribute to the persistent activated state of scar-forming fibroblasts. However, how epigenetic changes regulate fibroblast metabolic responses to promote fibroblast activation and progressive fibrosis remains largely unknown. Here, we show that the epigenetic regulator CBX5 (chromobox protein homolog 5) is critical to the transition of quiescent fibroblasts to activated collagen-producing fibroblasts in response to bleomycin-induced lung injury. Loss of mesenchymal CBX5 attenuated fibrosis development, and this effect was accompanied by the downregulation of pathogenic fibroblast genes, including Cthrc1, Col1a1, and Spp1, and by the upregulation of metabolic genes with antifibrotic activity such as Ppara and Pparg. Single-cell RNA sequencing and immunohistochemistry analyses revealed that CBX5 expression was enriched in pathogenic fibroblasts and fibroblastic foci of IPF lungs. Bulk RNA-sequencing analysis combined with metabolic assessments demonstrated that CBX5 silencing in IPF fibroblasts potently inhibited transforming growth factor–stimulated glycolysis while enhancing AMPK signaling and mitochondrial metabolism. Finally, interruption of the CBX5 pathway in IPF fibroblasts in vitro and in IPF lung explants ex vivo synergistically potentiated the activation of metformin-induced AMP-activated protein kinase activation and inhibited collagen secretion. Collectively, our findings identify CBX5 as an epigenetic regulator linking metabolic maladaptation to the persistent activated state of lung fibroblasts during IPF progression.