Last Update: August 2016
Extracellular Vesicles (EVs) have been linked to promising diagnostic, and therapeutic potentials, their characterisation, however, has been to date been poorly understood due to the lack of standardisation in detection equipment, and lack of resolution currently detection techniques hold. The term ‘extracellular vesicles’ encompasses a range of vesicles. These include apoptotic vesicles (AVs, apoptotic bodies and apoptotic blebs), microvesicles (MVs, microparticles, MPs, and ectosomes), exosomes and retrovirus-like vesicles (RLVs). Lipoproteins could also be considered within the umbrella term of EVs, due to being structurally similar (outer lipid membrane (monolayer), surface proteins, overlapping diameters) and possibly being mistaken for other EV types in current isolation and detection methods.
Discovery of EVs
The discovery of extracellular vesicle has spanned the 20th century. The first of this group to be discovered were lipoproteins, whose discovery spanned from the early 1920s to 1950. Apoptotic vesicles, RLVs, and MVs were identified in the 1960s, followed by exosomes in the 1980s. The vesicles contributing to this umbrella term are therefore not a recent discovery. The definitions of each of these different vesicles, however, remains problematic, due to the inconsistency in published data regarding the measurable criteria allowing their differentiation.
Microvesicle (MV) identification was a result of coagulation research spanning from the 1940s by Chargaff and West, until the late 1960s when Wolf reported visual evidence of platelet activation, using electron microscopy (4, 5). The activated platelets appeared to be shedding small vesicles (20- 50nm) directly from the plasma membrane, capable of thrombin generation. These vesicles were so minute in comparison to platelets that Wolf termed them ’platelet dust’. Wolf noted a linear correlation between the levels of platelet MVs (PMVs) and the original platelet count. He also noted that individuals with polycythaemia had a higher concentration of PMVs, with lower concentrations in individuals with thrombocytopenia.
Exosomes were notably identified in 1987 by Johnstone, who was researching the transformation process of reticulocytes into mature erythrocytes. Using electron microscopy, Johnstone identified small vesicles (30-100nm) held within the cytoplasm in multivesicular bodies (MVBs). Transferrin was identified as an abundant protein on MVBs using immunogold labelling, with the MVBs eventually binding to the plasma membrane, releasing the enclosed exosomes (6, 7).
Retrovirus-like Vesicles (RLVs) are thought to arise from human endogenous retrovirus (HERV) sequences, which contribute approximately 5-8% of the human genome, and were reported as early as 1965 (8, 9). RLVs are grouped into families denoted by a suffix letter i.e. HERV-A. HERV-K is the only one of these families that contain open reading frames for functional retroviral proteins, gag, env, rec and pol (10, 11). Cells experiencing stress, such as radiation, chemical treatment, cytokine stimulation, hormone stimulation, or oncogenic transformation can cause the usually repressed expression HERV-K genes to be de-repressed (12-19). RLVs bud directly from the plasma membrane and their mechanism of biogenesis is thought to differ from those of MVs or exosomes. Their diameter is currently being reported as fairly homogenous at 90-100nm (20).
Summary of EVs
Although each of these EVs differs, there is overlap in density, diameter, composition and derivation. Further study for more precise definitions and function, as well as the development of better identification machinery and markers, is therefore required. The main focus of the extracellular vesicle field is on the study of microvesicles and exosomes. Whilst lipoproteins, RLVs and apoptotic vesicles are not being highly studied in the EV field, they must be considered due to their overlapping properties, and for potentially causing contamination and inadvertent misinformation in publications to date due to their properties and the detection/isolation techniques used.
- Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26(4):239-57.
- Hristov M, Erl W, Linder S, Weber PC. Apoptotic bodies from endothelial cells enhance the number and initiate the differentiation of human endothelial progenitor cells in vitro. Blood. 2004;104(9):2761-6.
- Turiak L, Misjak P, Szabo TG, Aradi B, Paloczi K, Ozohanics O, et al. Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice. J Proteomics. 2011;74(10):2025-33.
- Thery C, Boussac M, Veron P, Ricciardi-Castagnoli P, Raposo G, Garin J, et al. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol. 2001;166(12):7309-18.
- Wolf P. The nature and significance of platelet products in human plasma. Br J Haematol. 1967;13(3):269-88.
- Chargaff E, West R. The biological significance of the thromboplastic protein of blood. J Biol Chem. 1946;166(1):189-97.
- Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol. 1985;101(3):942-8.
- Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem. 1987;262(19):9412-20.
- Belshaw R, Pereira V, Katzourakis A, Talbot G, Paces J, Burt A, et al. Long-term reinfection of the human genome by endogenous retroviruses. Proc Natl Acad Sci U S A. 2004;101(14):4894-9.
- Kajima M, Pollard M. Detection of viruslike particles in germ-free mice. J Bacteriol. 1965;90(5):1448-54.
- Bock M, Stoye JP. Endogenous retroviruses and the human germline. Curr Opin Genet Dev. 2000;10(6):651-5.
- Barbulescu M, Turner G, Seaman MI, Deinard AS, Kidd KK, Lenz J. Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans. Curr Biol. 1999;9(16):861-8.
- Florl AR, Lower R, Schmitz-Drager BJ, Schulz WA. DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas. Br J Cancer. 1999;80(9):1312-21.
- Gotzinger N, Sauter M, Roemer K, Mueller-Lantzsch N. Regulation of human endogenous retrovirus-K Gag expression in teratocarcinoma cell lines and human tumours. J Gen Virol. 1996;77 ( Pt 12):2983-90.
- Yoder JA, Walsh CP, Bestor TH. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 1997;13(8):335-40.
- Depil S, Roche C, Dussart P, Prin L. Expression of a human endogenous retrovirus, HERV-K, in the blood cells of leukemia patients. Leukemia. 2002;16(2):254-9.
- Reiche J, Pauli G, Ellerbrok H. Differential expression of human endogenous retrovirus K transcripts in primary human melanocytes and melanoma cell lines after UV irradiation. Melanoma Res. 2010;20(5):435-40.
- Golan M, Hizi A, Resau JH, Yaal-Hahoshen N, Reichman H, Keydar I, et al. Human endogenous retrovirus (HERV-K) reverse transcriptase as a breast cancer prognostic marker. Neoplasia. 2008;10(6):521- 33.
- Wang-Johanning F, Frost AR, Jian B, Epp L, Lu DW, Johanning GL. Quantitation of HERV-K env gene expression and splicing in human breast cancer. Oncogene. 2003;22(10):1528-35.
- Taruscio D, Mantovani A. Factors regulating endogenous retroviral sequences in human and mouse. Cytogenet Genome Res. 2004;105(2-4):351-62.
- Bieda K, Hoffmann A, Boller K. Phenotypic heterogeneity of human endogenous retrovirus particles produced by teratocarcinoma cell lines. J Gen Virol. 2001;82(Pt 3):591-6.
- van Meer G, Voelker DR, Feigenson GW. Membrane lipids: where they are and how they behave. Nature reviews Molecular cell biology. 2008;9(2):112-24.
Text modified from Welsh, Joshua (2016) Flow cytometer optimisation and standardisation for the study of extracellular vesicles as translational biomarkers University of Southampton Doctoral Thesis, 209pp.