Dataset Information

FACS-Proteomics strategy toward Extracellular Vesicles single-phenotype characterization in biological fluids: insights into the role of Leukocyte-derived EVs in Multiple Sclerosis

ABSTRACT: The isolation and proteomics characterization of Extracellular Vesicles (EVs) from body fluids represents a stimulating challenge to improve our knowledge of biology, considering the vast heterogeneity in terms of phenotype in vivo. Within a complex and dynamic scenario of EVs isolation and characterization protocols, we have recently demonstrated how the Fluorescence-activated Cell Sorting (FACS) strategy can be adopted to isolate total EVs from untouched body fluids enabling proteomics characterization. Here, we reported for the first time the proteomics characterization of EVs single-phenotype by sorting labelled-recognition events related to Leucocyte-derived EVs (Leuko EVs) by combining lipophilic dye/phalloidin and anti-CD45+ antibodies from peripheral blood and tears of enrolled volunteers. More than 90% of proteins identified in both biological fluids were mainly referred to leukocyte phenotype. This strategy was applied to carry out a label-free proteomics on Leuko EVs from tears of Multiple Sclerosis (MuS) patients, demonstrating as they are enriched with an encoded protein cargo, reflecting the neuroinflammatory condition of MuS (TGFB1 and NFE2L2, upstream regulators activated) and the endothelial cell proliferation together with an increase of vascular networks (AGNPT2 and VEGF, upstream regulators activated) which in turn reflect pro-angiogenic processes predicted as significantly up-regulated downstream effects. Our data showed that FACS-Proteomics strategy for EVs single phenotype characterization could have groundbreaking avenue for biomarker discovery, exalting the clinical value of tears EVs and helping in a better understanding of the EV-mediated processes in vivo.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Extracellular Exosome

DISEASE(S): Multiple Sclerosis

SUBMITTER: Maria Concetta Cufaro

LAB HEAD: Piero Del Boccio

PROVIDER: PXD049036 | Pride | 2025-05-26

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20220210_EVsLeuko_CTRL_02.raw	Raw
	20220210_EVsLeuko_CTRL_03.raw	Raw
	20220210_EVsLeuko_CTRL_04.raw	Raw
	20220210_EVsLeuko_SM_01.raw	Raw
	20220210_EVsLeuko_SM_03.raw	Raw

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Publications

FACS-Proteomics strategy toward extracellular vesicles single-phenotype characterization in biological fluids: exploring the role of leukocyte-derived EVs in multiple sclerosis.

Cufaro Maria Concetta MC Lanuti Paola P De Bellis Domenico D Veschi Serena S Piro Anna A Fontana Antonella A Di Sebastiano Alice A Brocco Davide D Simeone Pasquale P Pilato Serena S Khorooshi Reza M H RMH Tomassini Valentina V Rispoli Marianna Gabriella MG Federici Luca L Cicalini Ilaria I Pieragostino Damiana D Del Boccio Piero P

Journal of translational medicine 20250520 1

<h4>Background</h4>The isolation and proteomics characterization of extracellular vesicles (EVs) from body fluids is challenging due to their vast heterogeneity. We have recently demonstrated that Fluorescence-activated Cell Sorting (FACS) efficiently isolates the whole EV circulating compartment directly from untouched body fluids enabling a comprehensive EV proteomics analysis.<h4>Results</h4>Here, we characterized, for the first time, a single-phenotype EV subset by sorting leukocyte-derived ...[more]

PMID: 40394633

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

Project description:Sjogren’s syndrome (SS) is characterized by dysfunctional mucous membranes and dysregulated moisture-secreting glands resulting in various symptoms, including dry mouth and dry eyes. Here, we wanted to profile and compare the tears and saliva proteomes of SS patients to healthy controls. Tear and saliva samples were collected and subjected to an isotopic dimethylation labeling shotgun proteomics workflow to identify alterations in protein levels. In tear samples, we identified 83 upregulated and 112 downregulated proteins. Altered pathways in SS patients’ tears included leukocyte transendothelial migration, neutrophil degranulation, and post-translation protein phosphorylation. In healthy controls’ tears, enriched proteins for glycolysis, amino acid metabolism and apoptotic signaling pathway were identified. In saliva, we identified 108 upregulated and 45 downregulated proteins. Altered pathways in SS patients’ saliva included cornification, sensory perception to taste and neutrophil degranulation. In healthy controls’ saliva, enriched proteins for JAK-STAT signaling after interleukin-12 stimulation, phagocytosis and glycolysis in senescence were identified. Dysregulated protease activity is implicated in the initiation of inflammation and immune cell recruitment in SS. We identified 20 proteases and protease inhibitors in tears and 18 in saliva which are differentially expressed between SS patients and healthy controls. Next, we quantified endogenous proteoglycan 4 (PRG4), a mucin-like glycoprotein previously shown to be present in tears and never studied in saliva, in tear wash and saliva samples via a bead-based immune assay. We identified decreased levels of PRG4 in SS patients’ tear wash compared to normal samples. Conversely, in saliva, we found elevated levels of PRG4 concentration and visualized PRG4 expression in human parotid gland via immunohistological staining. These findings improve our mechanistic understanding of the disease and changes in SS patients’ protein expression will help identify new potential drug targets. PRG4 is among the promising targets, which we identified here, in saliva, for the first time.