Abstract
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, and highly fatal disease. It is characterized by the increased activation of both fibroblast and myofibroblast that results in excessive extracellular matrix (ECM) deposition. Extracellular vesicles (EVs) have been described as key mediators of intercellular communication in various pathologies. However, the role of EVs in the development of IPF remains poorly understood. This study aimed to characterize the differentially expressed proteins contained within EVs cargo derived from the fibroblast cell lines LL97A (IPF-1) and LL29 (IPF-2) isolated from lungs bearing IPF as compared to those derived from the fibroblast cell lines CCD8Lu (NL-1) and CCD19Lu (NL-2) isolated from healthy donors. Isolated EVs were subjected to label-free quantitative proteomic analysis by LC-MS/MS, and as a result, 331 proteins were identified. Differentially expressed proteins were obtained after the pairwise comparison, including all experimental groups. A total of 86 differentially expressed proteins were identified in either one or more comparison groups. Of note, proteins involved in fibrogenic processes, such as tenascin-c (TNC), insulin-like-growth-factor-binding protein 7 (IGFBP7), fibrillin-1 (FBN1), alpha-2 collagen chain (I) (COL1A2), alpha-1 collagen chain (I) (COL1A1), and lysyl oxidase homolog 1 (LOXL1), were identified in EVs cargo isolated from IPF cell lines. Additionally, KEGG pathway enrichment analysis revealed that differentially expressed proteins participate in focal adhesion, PI3K-Akt, and ECM–receptor interaction signaling pathways. In conclusion, our findings reveal that proteins contained within EVs cargo might play key roles during IPF pathogenesis.
Highlights
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible disease of somewhat uncertain etiology and pathogenesis [1,2]
Supernatants were purified from NL-1, NL-2, IPF-1, and IPF-2 cultured cell lines by differential centrifugation (Figure 1A)
Western blot analysis from Extracellular vesicles (EVs)-isolated proteins showed that extracts were highly enriched in Alix, HSP90, and Flotilin-1, three wellknown EVs markers (Figure 1B), indicating that samples obtained from the cell culture supernatants were enriched with proteins contained within EVs cargo
Summary
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible disease of somewhat uncertain etiology and pathogenesis [1,2]. Biomedicines 2021, 9, 1058 fibroblasts and myofibroblasts proliferation that results in exacerbated production and accumulation of extracellular matrix (ECM), promoting pulmonary parenchyma remodeling, which in turn leads to pulmonary insufficiency, and eventually, patient death [2]. Some genetic variants have been strongly associated with IPF development [3,4]. Variants of the MUC5B gene, encoding for mucin 5B protein, are associated with the maintenance of bronchoalveolar epithelial function [3,4,5]. Variants of TERT and TERC genes, which encode for telomerase reverse transcriptase, and the RNA component of telomerase, respectively, associated with the telomere length maintenance, telomere shortening is a feature that has been associated with the development of IPF [3,4]
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