Dataset Information

CNS-EVs enriched by PROSPR - Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR

ABSTRACT: Study of EVs from CNS tissues becomes challenging with the current available methods of EVs enrichment. We recently developed PROSPR, a standardized and easy-to-perform method to enrich EVs from biological fluids. We have optimized the use of PROSPR to efficiently enrich EVs from CNS tissues.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Cerebral Cortex

SUBMITTER: Xavier Gallart Palau

LAB HEAD: Siu Kwan Sze

PROVIDER: PXD003288 | Pride | 2016-05-31

REPOSITORIES: Pride

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Source:

			Action	DRS
	151002_PROSPR_Brain_EVs_01_A.raw	Raw
	151002_PROSPR_Brain_EVs_01_B.raw	Raw
	151002_PROSPR_Brain_EVs_01_C.raw	Raw
	151002_PROSPR_Brain_EVs_02_A.raw	Raw
	151002_PROSPR_Brain_EVs_02_B.raw	Raw

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Enrichment of extracellular vesicles from tissues of the central nervous system by PROSPR.

Gallart-Palau Xavier X Serra Aida A Sze Siu Kwan SK

Molecular neurodegeneration 20160523 1

<h4>Background</h4>Extracellular vesicles (EVs) act as key mediators of intercellular communication and are secreted and taken up by all cell types in the central nervous system (CNS). While detailed study of EV-based signaling is likely to significantly advance our understanding of human neurobiology, the technical challenges of isolating EVs from CNS tissues have limited their characterization using 'omics' technologies. We therefore developed a new Protein Organic Solvent Precipitation (PROSP ...[more]

PMID: 27216497

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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.