Proteome Characterization of BALF Extracellular Vesicles in Idiopathic Pulmonary Fibrosis: Unveiling Undercover Molecular Pathways.

Enxhi Shaba,Pietro Lupetti,Elena Bargagli,Eugenio Paccagnini,Alfonso Carleo,Laura Bianchi,Mariangela Gentile,Claudia Landi,Lorenza Vantaggiato,Luca Bini,Antje Prasse

doi:10.3390/ijms22115696

Abstract

In the longtime challenge of identifying specific, easily detectable and reliable biomarkers of IPF, BALF proteomics is providing interesting new insights into its pathogenesis. To the best of our knowledge, the present study is the first shotgun proteomic investigation of EVs isolated from BALF of IPF patients. Our main aim was to characterize the proteome of the vesicular component of BALF and to explore its individual impact on the pathogenesis of IPF. To this purpose, ultracentrifugation was chosen as the EVs isolation technique, and their purification was assessed by TEM, 2DE and LC-MS/MS. Our 2DE data and scatter plots showed considerable differences between the proteome of EVs and that of whole BALF and of its fluid component. Analysis of protein content and protein functions evidenced that EV proteins are predominantly involved in cytoskeleton remodeling, adenosine signaling, adrenergic signaling, C-peptide signaling and lipid metabolism. Our findings may suggest a wider system involvement in the disease pathogenesis and support the importance of pre-fractioning of complex samples, such as BALF, in order to let low-abundant proteins-mediated pathways emerge.

Highlights

Bronchoalveolar lavage (BAL) is a relatively non-invasive procedure used in pulmonary medicine, consisting of washing selected lobes of the lung with saline buffer solution by fiberoptic bronchoscopy and in the recovery of the fluid
extracellular vesicles (EVs) were isolated from Bronchoalveolar Lavage Fluid (BALF) of IPF patients (Table 1) by sequential ultracentrifugation, and their purification was assessed by Transmission Electron Microscopy (TEM) (Figure 1)
Images of EVs from BALF of IPF patients (Figure 1a), this reproducible isolation technique enabled the separation of a wide size range (40–2000 nm) of vesicles with spherical morphology, sometimes assembled into small aggregates

Summary

Introduction

Bronchoalveolar lavage (BAL) is a relatively non-invasive procedure used in pulmonary medicine, consisting of washing selected lobes of the lung with saline buffer solution by fiberoptic bronchoscopy and in the recovery of the fluid. This fluid consists mainly of cells, both resident alveolar cells and recruited inflammatory cells, their secreted products and proteins leaked across the endothelial–epithelial barrier. Its cell-free component, commonly referred to as “Bronchoalveolar Lavage Fluid (BALF),” is quite similar in composition to other biological fluids, especially plasma, consisting mainly of phospholipids, lipids, nucleic acids, peptides and proteins derived from resident cells and/or passive/active diffusion through the alveolar–capillary barrier [1,2]. BALF examination is, an optimal tool of ELF assessment and of diagnosis and monitoring of pulmonary diseases [3]

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