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. 2012 Apr 16;1.
doi: 10.3402/jev.v1i0.18396. eCollection 2012.

Cellular Stress Conditions Are Reflected in the Protein and RNA Content of Endothelial Cell-Derived Exosomes

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Free PMC article

Cellular Stress Conditions Are Reflected in the Protein and RNA Content of Endothelial Cell-Derived Exosomes

Olivier G de Jong et al. J Extracell Vesicles. .
Free PMC article

Abstract

Background: The healthy vascular endothelium, which forms the barrier between blood and the surrounding tissues, is known to efficiently respond to stress signals like hypoxia and inflammation by adaptation of cellular physiology and the secretion of (soluble) growth factors and cytokines. Exosomes are potent mediators of intercellular communication. Their content consists of RNA and proteins from the cell of origin, and thus depends on the condition of these cells at the time of exosome biogenesis. It has been suggested that exosomes protect their target cells from cellular stress through the transfer of RNA and proteins. We hypothesized that endothelium-derived exosomes are involved in the endothelial response to cellular stress, and that exosome RNA and protein content reflect the effects of cellular stress induced by hypoxia, inflammation or hyperglycemia.

Methods: We exposed cultured endothelial cells to different types of cellular stress (hypoxia, TNF-α-induced activation, high glucose and mannose concentrations) and compared mRNA and protein content of exosomes produced by these cells by microarray analysis and a quantitative proteomics approach.

Results: We identified 1,354 proteins and 1,992 mRNAs in endothelial cell-derived exosomes. Several proteins and mRNAs showed altered abundances after exposure of their producing cells to cellular stress, which were confirmed by immunoblot or qPCR analysis.

Conclusion: Our data show that hypoxia and endothelial activation are reflected in RNA and protein exosome composition, and that exposure to high sugar concentrations alters exosome protein composition only to a minor extend, and does not affect exosome RNA composition.

Keywords: RNA; exosomes; extracellular vesicles; hypoxia; proteomics; tumor necrosis factor alpha.

Figures

Fig. 1
Fig. 1
Characterization of endothelial cell-derived exosomes. Vesicles in the final, washed 100,000g pellets from endothelial cell culture supernatant cultured under standard conditions were analysed by sucrose density gradient and subsequent immunoblotting for Flotillin-1 (a), transmission electron microscopy (b; 10 nm gold: CD63) and by NTA (c). Size distributions of vesicles in washed 100,000g pellets from supernatants of stress-exposed endothelial cells were compared by NTA (d–g; conditions indicated, grey area represents SD).
Fig. 2
Fig. 2
Verification of quantitative proteomics data by immunoblotting. Differences observed in quantitative proteomics, expressed as mean±SD based on analysis of individual quantified peptides (a–c) were verified by immunoblotting (d–f).
Fig. 3
Fig. 3
Analysis and verification of exosome mRNA profiles. Quantitative PCR confirms differential abundance of mRNAs identified to be significantly increased or decreased in exosomes from hypoxia (a) or TNF-α exposed cells compared to those from control cells (b), error bars represent SEM, *p < 0.05. Clustering analysis based on the 100 most variable mRNAs distinguishes exosomes from endothelial cells exposed to hypoxia or TNF-α, but not to high glucose or mannose, from control cell-derived exosomes (c).

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References

    1. Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G. The biogenesis and functions of exosomes. Traffic. 2002;3:321–30. - PubMed
    1. Poliakov A, Spilman M, Dokland T, Amling CL, Mobley JA. Structural heterogeneity and protein composition of exosome-like vesicles (prostasomes) in human semen. Prostate. 2009;69:159–167. - PubMed
    1. Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2:569–610. - PubMed
    1. Fevrier B, Raposo G. Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr Opin Cell Biol. 2004;16:415–21. - PubMed
    1. Ohshima K, Inoue K, Fujiwara A, Hatekeyama K, Kanto K, Watanabe Y, et al. Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line. PLoS One. 2010;5:e13247. - PMC - PubMed

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