The profile of miRNAs in lung fibroblast-derived extracellular vesicles from idiopathic pulmonary fibrosis (IPF) patients or non-IPF individuals.. The profile of miRNAs in lung fibroblast-derived extracellular vesicles from idiopathic pulmonary fibrosis (IPF) patients or non-IPF individuals.

Accelerated senescence in lung epithelial cells is known to play a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, the exact mechanisms underlying the IPF-related epithelial cell phenotype have yet to be elucidated. Increasing evidence supports the concept that extracellular vesicles (EVs), including exosomes and microvesicles, mediate intercellular communication that contributes to diverse aspects of physiology and pathogenesis. Here, we demonstrate that lung fibroblasts (LFs) from IPF patients accelerate epithelial cell senescence via EV-mediated transfer of LF-derived pathogenic cargo to lung epithelial cells. Mechanistically, IPF LF-derived EVs increase mitochondrial reactive oxygen species (mtROS) and associated mitochondrial damage in lung epithelial cells, leading to mtROS-mediated activation of the DNA damage response and subsequent epithelial cell senescence. We show that IPF LF-derived EVs contain elevated levels of miR-23b-3p and miR-494-3p that are responsible for suppressing SIRT3, resulting in the EV-induced phenotypic changes of lung epithelial cells. Furthermore, we observe that miR-23b-3p and miR-494-3p expression increases in lung epithelial cells from IPF patients’ lungs. Finally, the levels of miR-23b-3p and 494-3p found in IPF LF-derived EVs correlate positively with IPF disease severity. These findings reveal that the accelerated epithelial cell mitochondrial damage and senescence observed during IPF pathogenesis are caused by a novel mechanism in which SIRT3 is suppressed by miR-containing EVs derived from IPF fibroblasts. Overall design: We pooled six of the individual IPF LF-derived EV samples and six of the individual non-IPF LF-derived EV samples. Individual EV sample was isolated from the conditioned media of primary lung fibroblasts by ultratrancefugation. Total RNAs were extracted from these EVs. Microarray analysis was performed using 20ng of the RNAs.

已知肺上皮细胞的加速衰老在特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）的发病机制中发挥关键作用。然而，与IPF相关的上皮细胞表型的确切调控机制尚未阐明。越来越多的证据表明，包括外泌体（exosomes）与微囊泡（microvesicles）在内的细胞外囊泡（extracellular vesicles, EVs）可介导细胞间通讯，该过程参与生理活动与疾病发病的多个环节。本研究证实，IPF患者来源的肺成纤维细胞（lung fibroblasts, LFs）可通过EV介导的肺成纤维细胞来源致病载荷向肺上皮细胞的转运，加速上皮细胞衰老。从机制层面分析，IPF肺成纤维细胞来源的EVs可上调肺上皮细胞内的线粒体活性氧（mitochondrial reactive oxygen species, mtROS）水平并诱发相关线粒体损伤，进而触发mtROS介导的DNA损伤应答激活，最终导致上皮细胞衰老。本研究显示，IPF肺成纤维细胞来源的EVs中miR-23b-3p与miR-494-3p的表达水平显著升高，这两种微小RNA可通过抑制沉默信息调节因子3（SIRT3），介导EV诱导的肺上皮细胞表型改变。进一步研究发现，IPF患者肺组织的肺上皮细胞内miR-23b-3p与miR-494-3p的表达水平同样升高。最终，IPF肺成纤维细胞来源的EVs中miR-23b-3p与miR-494-3p的水平与IPF疾病严重程度呈显著正相关。本研究结果揭示，IPF发病过程中观察到的上皮细胞线粒体损伤加速与衰老现象，由一种全新机制介导：IPF成纤维细胞来源的含微小RNA的EVs可抑制SIRT3的表达。整体实验设计：我们将6份独立的IPF肺成纤维细胞来源EV样本与6份独立的非IPF肺成纤维细胞来源EV样本分别进行混合。每份独立EV样本均通过超速离心法从原代肺成纤维细胞的条件培养基中分离获得。从上述EV样本中提取总RNA，取20ng RNA进行基因芯片分析。