Functional proteomic investigation of extracellular vesicles: Rai +/+ vs Rai -/- astrocytes and released vesicles in EAE and bronchoalveolar lavage fluid-extracted extracellular vesicles in idiopathic pulmonary fibrosis

In the recent years, extracellular vesicles (EVs) drew a growing interest in scientific community, especially for their intrinsic role of cell-cell communication entities between local and distant targets. Given these appealing features, functional proteomics of EVs is a considerably valuable approach for the investigation of not only their functions, but also of their potentialities, as it provides a wider and enriched scenario of various physio-pathological molecular mechanisms. Considering this background, we performed two different functional proteomic studies on EVs in two different pathologic conditions. The first study emerged from recent investigations on protein ShcC/Rai role in experimental autoimmune encephalomyelitis (EAE), as its deficiency resulted in disease protection and astrocytes were identified as accountable for the establishment of a local protective environment. Therefore, our analysis focused on the differences in protein content of astrocytes, as well as of their released extracellular vesicles, between Rai+/+ and Rai -/- in a not stimulated and IL-17-stimulated conditions, applying 2DE, image analysis, mass spectrometry identification of differential proteins and enrichment analysis. Curiously, our proteomic data showed that both the vesicular and cellular differential proteins indicate the overall involvement of macro molecular areas, such as oxidative stress response, ECM and cellular remodelling, glutamate metabolism, EMT mechanisms and metabolic reprogramming, shifting astrocytes towards a neuroprotective response. Concurrently, a second study was conducted to characterize and explore the individual impact on Idiopathic Pulmonary Fibrosis (IPF) pathogenesis of not only the vesicular component of bronchoalveolar lavage fluid (BALF), but also its fluid counterpart. Indeed, to the best of our knowledge, our study is the first shotgun proteomic investigation of EV isolated from BALF of IPF patients. To this purpose, ultracentrifugation was chosen as EVs isolation technique and its purification was assessed by TEM, 2DE and LC-MS/MS. Interestingly, our 2DE data and scatter plot analysis showed a considerable difference of EVs proteome with respect to whole BALF and to its fluid counterpart proteome, highlighting the importance of pre-fractioning of complex samples to the advantage of low-abundant protein species in biomarkers discovery. Remarkably, enrichment analysis results draw attention on a systemic metabolic dysregulation in disease development and highlight relevant molecular pathways that result distinctive but complementary in IPF pathogenesis.