Dataset Information

Cigarette smoke extract-treated alveolar macrophage-derived extracellular vesicles

ABSTRACT: This project is based upon the fundamental observation that alveolar macrophage-derived extracellular vesicles (AM-EVs), when internalized by neighboring epithelial cells, inhibit their infection by influenza virus. This inhibitory activity of AM-EVs is abolished when AMs are treated with cigarette smoke extract (CSE). We chose to survey the AM-EV proteome in an effort to identify candidate proteins whose abundance within EVs was downregulated by CSE treatment of AMs, thus explaining the ability of CSE to abrogate the inhibitory activity against influenza.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Lung, Cell Culture, Macrophage

DISEASE(S): Disease Free

SUBMITTER: Daniel Schneider

LAB HEAD: Daniel Joseph Schneider

PROVIDER: PXD019528 | Pride | 2020-06-09

REPOSITORIES: Pride

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Source:

			Action	DRS
	PRF_F_2019_M_PETE_81_1.raw	Raw
	PRF_F_2019_M_PETE_81_1_8.msf	Msf
	PRF_F_2019_M_PETE_81_1_8.msfView	Msf
	PRF_F_2019_M_PETE_81_1_8.pdResult	Other
	PRF_F_2019_M_PETE_81_1_8.pdResultView	Other

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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:Introduction: Endometriosis is a debilitating condition where endometrial-like tissue grows outside the uterus and rarely in distant sites such as the lungs, heart and brain. Symptoms are severe chronic pain, bladder/bowel problems, painful sex and infertility. Differences in endometrium between women with and without endometriosis have not been constructive, and there is still paucity in its pathogenesis and non-invasive diagnostic test, leading to delayed diagnosis (~10 years) and effective treatment. Menstrual fluid (MF) gives a peak into endometrial health. Could MF extracellular vesicles (EVs) point to the pathogenesis and a non-invasive diagnostic tool for endometriosis? Aims: To identify differences in protein-cargo in MF-EVs from women with and without endometriosis, define their functional contribution to mesothelial niche cells for lesion establishment and determine whether MF-sEV can be a non-invasive endometriosis diagnostic tool. Methods: MF was collected from women with and without endometriosis on day 2 of menses and EVs isolated using differential centrifugation. MF-EVs were identified by size, morphology and protein markers and subjected to TMT- quantitative proteomics assay. Differentially expressed proteins were analysed. Ctrl and Endo MF-sEV were co-cultured with mesothelial cells to assess for uptake and changes in the adhesive and junctional proteins. Results: Spherical-shaped MF-EVs were identified with mean diameters of 121.9 nm (Endo) and 123.8 nm (Ctrl), expressing TSG101, Alix and Syntenin-1 sEV proteins. MF-sEV proteins originated from endometrial cells: stem cells, leucocytes, and endothelial and smooth muscle cells, validating the endometrial origin of the MF-EVs. 77% of identified proteins were differentially expressed. In endo-MF-sEV, proteins regulating HGF receptor (anoikis), cellular senescence and multiple apoptotic, nitrogen compound metabolic processes and TRP channels signalling (immunity and contraction regulation) were decreased, potentially promoting lesion establishment, survival and pain. Similarly, antibacterial peptides (cathelicidin, mucin-1, lipocalin2 and serum amyloid A) were downregulated. The majority of proteins were significantly downregulated in endometriosis suggesting a dysregulation in the protein synthesis and cellular communication for homeostasis. Two-thirds of upregulated proteins were immunoglobulins indicating inflammatory pathogenesis. This was reflected by an increased Endo-MF-EVs uptake by mesothelial cells, with decreased scribble resulting in decreased cellular resistance and permeability. Conclusions: MF-EVs contain vital information indicative of endometriosis pathogenesis and may be the key to developing a simple and early diagnostic tool.

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Cigarette smoke extract-treated alveolar macrophage-derived extracellular vesicles

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