Dataset Information

Human and mouse brain, and human cerebrospinal fluid extracellular vesicles LC-MSMS

ABSTRACT: Extracellular vesicles (EVs) are a unified terminology of membrane-enclosed vesicular species ubiquitously secreted by almost every cell type and present in all body fluids. They carry a cargo of lipids, metabolites, nucleic acids and proteins for their clearance from cells as well as for cell-to-cell communications. The exact composition of EVs and their specific functions are not well understood due to the underdevelopment of the separation protocols, especially those from the central nervous system including animal and human brain tissues as well as cerebrospinal fluids. To understand their exact molecular composition and their functional roles, development of the reliable protocols for EV separation is necessary. Known EV separation methods include differential centrifugation-ultracentrifugation, sucrose gradient ultracentrifugation, size exclusion chromatography, ultra-filtration, microfluidics, high-resolution flow cytometric sorting, polymer-based precipitation, immunoaffinity capture, affinity capture, and asymmetric-flow field-flow fractionation. In this report, we describe our EV separation protocols from human biospecimens, such as unfixed frozen brain tissues and cerebrospinal fluids. We also discuss the quality control measures of the above separation protocols by biophysical and proteomic characterizations.

INSTRUMENT(S): Q Exactive HF

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

TISSUE(S): Brain, Cerebrospinal Fluid

SUBMITTER: Satoshi Muraoka

LAB HEAD: Tsuneya Ikezu

PROVIDER: PXD017273 | Pride | 2020-02-17

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	HumanCSF-derivedEV.mzXML	Mzxml
	HumanCSF-derivedEV.raw	Raw
	Humanbrain-derivedEV_SG-UC.mzXML	Mzxml
	Humanbrain-derivedEV_SG-UC.raw	Raw
	Mousebrain-derivedEV_SG-UC.raw	Raw

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Publications

Assessment of separation methods for extracellular vesicles from human and mouse brain tissues and human cerebrospinal fluids.

Muraoka Satoshi S Lin Weiwei W Chen Mei M Hersh Samuel W SW Emili Andrew A Xia Weiming W Ikezu Tsuneya T

Methods (San Diego, Calif.) 20200205

Extracellular vesicle (EV) is a unified terminology of membrane-enclosed vesicular species ubiquitously secreted by almost every cell type and present in all body fluids. They carry a cargo of lipids, metabolites, nucleic acids and proteins for their clearance from cells as well as for cell-to-cell communications. The exact composition of EVs and their specific functions are not well understood due to the underdevelopment of the separation protocols, especially those from the central nervous sys ...[more]

PMID: 32035230

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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.