© Borgis - Postępy Nauk Medycznych 2/2016, s. 132-136
Możliwe znaczenie diagnostyczne mikrocząstek błon komórkowych
Possible diagnostic role of cell membrane microparticles
Department of Immunohaematology, Centre of Postgraduate Medical Education, Warsaw
Head of Department: Jadwiga Fabijańska-Mitek, PhD, Associate Professor
Mikropęcherzyki (egzosomy i ektosomy) są heterogenną grupą sferycznych błonowych struktur uwalnianych przez większość komórek. Wydzielane są z błon komórkowych podczas dojrzewania i starzenia komórek. Te struktury błonowe wykrywane są m.in. w moczu, we krwi, w płynie owodniowym i ślinie. Aktywacja komórek indukowana przez różne czynniki powoduje wzrost wydzielania pęcherzyków. Funkcja tych struktur, skład i specyficzna ekspresja markerów powierzchniowych zależą od typu komórki, z której powstają. Artykuł przedstawia najnowszy stan wiedzy dotyczącej ogólnej charakterystyki, klasyfikacji, metod badawczych i roli struktur błonowych w różnych chorobach. Mikropęcherzyki jako biomarkery, czynniki pośredniczące w oddziaływaniach międzykomórkowych oraz nośniki białek mogą mieć zastosowanie w diagnostyce i prognozowaniu wielu chorób, np.: choroby sercowo-naczyniowe, autoimmunologiczne, cukrzyca, zakażenia i progresja nowotworowa. Określenie pochodzenia i liczby krążących mikropęcherzyków ułatwia wyjaśnienie ich udziału w patogenezie różnych schorzeń. Wyjaśnienie biologii oraz powstawania pęcherzyków jest istotne w zrozumieniu ich roli w warunkach prawidłowych oraz w rozmaitych chorobach. Pomimo stosowanych kilku metod (cytometria przepływowa, mikroskopia, ELISA), dokładny mechanizm, jak również czynniki biorące udział w tworzeniu pęcherzyków są tylko częściowo wyjaśnione i wymagają dalszych badań.
Extracellular vesicles (exosomes and ectosomes) are heterogenous group of spherical membrane structures released by most cells. They are secreted from the cell membranes during maturation and aging of cells. These membrane structures were detected, among others, in urine, blood, amniotic fluid and saliva. Cell activation, induced by various factors results in increased secretion of vesicles. These structures function, composition and their specific surface markers expression, depend on the cell type origin, from which they are formed. The article presents an overview of current knowledge on vesicle general characteristics, classification, research methods and role of membrane structures in various diseases. Microvesicles as biomarkers, mediators in cell interactions and proteins transporters can be applied in the diagnostics and prognosis of many diseases including cardiovascular, autoimmune, diabetes, infectious diseases and cancer metastasis. Determining the origin and the number of circulating microparticles facilitates explanation of their participation in the pathogenesis of various disorders. The elucidation of the biology and vesicles formation is important for understanding of their role in health and various disorders. Despite different applied methods (flow cytometry, microscopy, ELISA), the exact mechanism and components of the vesicle involved process formation remain only partially established and requires further studies.
General characteristics of vesicles
Extracellular vesicles (EV) are spherical membrane structures released by different types of normal and also cancer cells (1). The best known example are the vesicles derived from blood platelets (2). These membrane structures were detected, among others, in urine, blood, amniotic fluid, saliva, or semen. Under physiological conditions they are released from the cell membranes during maturation and aging of cells. Cell activation, induced by various factors (complement proteins, cytokines, stress factors), results in increased secretion of these structures. Recently involvement of vesicles in many diseases, including cardiovascular, autoimmune, diabetes, infectious diseases and cancer metastasis was studied (3). Determining the origin and the number of circulating microparticles facilitates explanation of their participation in the pathogenesis of various disorders. A few publications have revealed an increased release of these structures during long-term storage of blood for transfusions (4-8).
History of vesicles studies
First report concerning the vesicles was published in 1967 and has been described by Wolf. The author termed the structures “platelet dust”, the presence of which he found in human blood plasma. Structures secreted by activated blood platelets showed prothrombotic properties, owing to the presence of tissue factor – thrombospondin (9). The vesicles were isolated by ultracentrifugation, and observed by the electron microscopy. Discovery of the “platelet dust” marked the beginning of a new era regarding microvesicles formation and their role in the organism. Afterwards first publications on microvesicles released from erythrocytes (10), monocytes (11) and endothelial cells were published (12).
The membrane vesicles are a heterogeneous group of different sizes, origin, biological and physical properties, mechanism and source of formation. Due to the size and origin, the vesicles were divided into: smaller exosomes, with a diameter of 30-100 nm, and bigger, ectosomes, measuring from 0.1 to 1 μm (13). Previous nomenclature of these structures was based on the origin of the cells, from which they are formed. In recent years terminology was based on the mechanism of their formation. Consensus on terminology and classification of vesicles was obtained in 2013 (14). Among the vesicles, the exosomes generated within the multivesicular bodies (MVBs) were separated. During the fusion of MVBs with the intraluminal membrane, vesicles are secreted from cells. After that, their size ranges from 40 to 150 nm. Major markers are tetraspanins (CD9, CD63, CD81, CD82), and markers of lipid rafts: flotillins 1 and 2. Other markers, such as heat shock proteins (HSP), MHC molecules, various components of endosomal sorting complex required for transport (ESCRT) and member of the Ras superfamily of monomeric G proteins (Rab) are used to identify these vesicles (15, 16).
The exosomes are formed and maturate in the intracellular (endosomal) vesicles. Afterwards they are secreted out of the cell by the fusion of vesicle membrane with the cell membrane, what involves soluble N-ethylmaleimide – sensitive factor attachment receptor transmembrane proteins (SNARE). In the process of exosome formation, Ca2+ dependent, vesicle associated membrane proteins (VAMP) are included. It was shown that in leukemia cells, the VAMP-7 protein plays an important role in the formation of exosomes during their fusion with the cell membrane. VAMP-1,-2,-3 proteins were identified in the exosomes secreted from tumor cells (13). Recent studies have shown that there are 2 types of exosomes: immunologically active exosomes that are involved in antigen presentation and costimulation and those containing RNA and mediating the genetic communication between cells (17).
Ectosomes are formed extracellularly, directly from the cell membrane by blebbing. The vesicle detachment occurs due to the contraction of cell, and then peeling it in specific sites. On the cell surface, the buds are formed, which mechanism involves active participation of cytoskeleton proteins e.g. actin. In the cell membrane proteins and lipids many changes take place. Similarly to the apoptosis, disruption of phospholipid asymmetry appears. In the cell membrane movement of phospholipids: phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and sphingomyelin takes place with the participation of flippase, floppase and scramblase. Ca2+ ions play an important role during the formation of microvesicles. As a result, the increased concentration of Ca2+ activates enzymes, such as gelsolin, aminophospholipid translocase, floppase, scramblase and calpain, resulting in the change of cell membrane asymmetry (18). Exposure of phosphatidylserine on the outside layer of the plasma membrane seems to be one of the main microvesicle properties, although there are also microvesicles without exposed phosphatidylserine. RhoA protein, belonging to the family of small guanosine-5’-triphosphates (GTPases), Rho-activated protein kinases (ROCK) and actin-binding (LIM) kinases are regulators of ectosome secretion. Other regulator, is calpain – Ca2+ dependent enzyme, which participates in the regulation of cytoskeleton proteins, where it plays a significant role in the ectosome formation. Furthermore it was also concluded that the ADP-ribosylation factor 6 (ARF6) protein is essential both in these structure formation and secretion (13).
Vesicles express different characteristic surface molecules depending on their cellular origin. Specific markers to identify structures are listed in table 1 (19, 20).
Table 1. Specific markers of vesicles releasing from different types of cells (by 19, 20, modified)
|MP origin||Common name||CD||Role|
|Erythrocytes||Glycophorin A||CD235a||Blood group antigen|
|Leucocytes||LCA (T200, B220)||CD45||Tyrosine phosphatase|
|Monocytes||LPS-R||CD14||LPS – binding protein|
|Cell adhesion, migration, pathogen binding, tumor progression|
|Th lymphocytes||T4||CD4||Th costimulation|
|Ts lymphocytes||T8 (Leu-2)||CD8||Tc costimulation|
|B lymphocytes||B1 (Bp35)||CD20||Regulation of B lymphocytes activation and proliferation|
|Platelets||GPIbα (β3 integrin)|
GPIIb (αIIβ integrin)
GPIIIa (β3 integrin)
|Adhesion and platelets aggregation|
Platelets aggregation, bind with CD61 molecule
Adhesion and platelets aggregation
Create αIIbβ3 and αvβ3 integrins
Leucocytes adhesion to endothelium
Leucocytes adhesion to endothelium
Transition into blood vessels
Cell adhesion, endothelium marker
Receptor for TGF-β1 and TGF-β3
A variety of research methods are used to study microvesicles: ELISA, flow cytometry, electron microscopy, atomic force microscopy (AFM), spectroscopy and nanoparticles tracking analysis (NTA). While examining the structures, during the isolation and preparation, special care should be taken in the sampling procedure, and suitable type of the anticoagulant has to be used. Sodium citrate is the best anticoagulant for vesicles preparation because it in the smallest extent activates the cells in comparison with other reagents. It is also known that the number of extracellular vesicles increases under the influence of stress factors. Therefore, during the preparation a special attention should be paid to the temperature changes and gentle mixing of samples. The time between sample collection and isolation should be as short as possible (21). Flow cytometry is preferably used for the evaluation of number and determination of vesicle sizes (22, 23). This technique was used for the first time to study these structures in 1991 (24). Flow cytometry studies allow evaluation of circulating vesicles with using the antibodies directed against the surface markers. Cytometric method allows to obtain information on the morphology (size and granularity) of these structures as evidenced by the parameters: FSC (Forward Scatter Channel) and SSC (Side Scatter Channel) (fig. 1). In order to separate and determine structures in a proper way, calibration beads of appropriate diameter are used (25, 26).
Fig. 1. Representative “dot-plot” obtained by flow cytometry. Determination of vesicles size. Flow cytometry analysis: calibration beads (size: 1, 2, 4, 6 μm), erythrocytes (7 μm) were used as an internal standard
During the formation of extracellular vesicles exposure of phosphatidylserine (PS) takes place, therefore annexin V, which binds to PS, is applied for staining. Additionally, antibodies against specific various cell markers should be used, since the vesicles are released from various cells. Proper separation, identification and evaluation of these structures is complicated and has many problems. Methods used for the isolation, qualitative and quantitative assessment are not standardized and create many technical issues. Key limitation in these studies is the lack of an internal validation process (27).
Another method of vesicle detection is Nanoparticle tracking analysis (NTA) consists of measuring and visualizating particles in liquid. On the basis of the velocity measurement of the Brownian motion, which moves particles, the size of flowing molecules can be determined (28-30). This method allows determining the size distribution profile of the structures size, measuring 10-1000 nm in suspension. Some authors consider that defining the vesicles size by the cytometric method using calibration beads is inappropriate. The beads have various optical parameters: optical density, diffraction coefficient and light absorption. On the other hand NTA allows to define vesicle sizes without influence of molecule density or refractive index. The speed of particle movement is only influenced by temperature and viscosity of the fluid.
Role of vesicles in various diseases
Increasing interest in potential usage of vesicles, as biomarkers of disorders, is related to their role in the diagnostics of many diseases. Increase of the circulating vesicles number, their composition and shape changes may reflect the pathophysiological conditions of many different disorders. Their role has been recognized in, among others, in cardiovascular, autoimmune diseases, disorders of the blood coagulation, diabetes and during cancer metastasis.
Different patterns of cellular vesicles in type 1 and 2 diabetic patients were observed. Increase in the extracellular vesicles being released from platelets, monocytes, lymphocytes, granulocytes and endothelial cells was detected. These structures can cause diabetic complications: vascular thrombosis, vascular inflammation, and angiogenesis. Clinical studies indicated that level of endothelial and platelet-derived vesicles is significantly increased in T1 diabetes (31). Omoto et al. (32) observed increase in the number of vesicles released from monocytes and platelets in T2DM patients. Moreover the elevated endothelial and erythrocytes-derived vesicle number was estimated in T2 diabetes (33, 34).
In cardiovascular diseases changes of endothelial and circulating cells-derived vesicles were also observed. Correlation between endothelial vesicles level and the severity of coronary artery disease, heart attack and brain stroke was detected.
Role of these structures during the tumor progression was analyzed by the scientists at the Canadian University. Studies were conducted on the glioblastoma multiforme cells, and vesicles were released from these cells termed oncosomes. In the oncosomes, the molecules of the epidermal growth factor receptor III (EGFR III) and epidermal growth factor (EGF) were identified that stimulate uncontrolled cell divisions. Vesicles float through the blood, migrate and attach to a normal cells or these in the early stage of cancer. Structures fuse with the normal cells, providing the receptor. Additional molecules of EGFRvIII stimulate excessive cell proliferation leading to the cancer progression (35). Furthermore, the oncosomes can also transfer mRNA, microRNA what involves the phenotype change of the normal cell.
Vesicles derived from blood cells may play role in the transfusion medicine. Several observations have shown a link between elevated number of vesicles released from blood cells during storage of blood concentrates and post-transfusion complications (36-38). During early blood banking, RBC vesicles formation and membrane remodeling was observed (6, 39). The exposure of phosphatidylserine on the outside layer of cell membrane takes place (39, 40). Level of RBC-derived vesicles gradually increases with storage time, as already observed in transfused patients with paroxysmal nocturnal haemoglobinuria (41). However, level of vesicle formation in erythrocyte concentrates depends not only on storage period but also on preparation and the storage solution (41-44). Structure of vesicles in erythrocytes concentrates is similar to those released in vivo. However differences in the levels of some proteins, e.g. stomatin (elevated level in membrane of RBCs vesicles in concentrates), integral membrane or cytoskeleton proteins were detected. Vesicles found in concentrates are devoid of most of these proteins (3, 6, 10). Vesicles in erythrocyte concentrates are more heterogeneous, with increase of size and content of components of proteasome.
Despite many methods, e.g. flow cytometry, proteomics, microscopy, ELISA, nanoparticle tracking analysis, spectroscopy, the exact and complete mechanism and components of the vesicle involved process formation remain only partially established. Understanding the microvesicle biology, quantitative and qualitative analysis could be one of the links useful to improve the diagnosis and prognosis of various diseases.
1. Chen J, Li C, Chen L: The role of microvesicles derived from mesenchymal stem cells in lung diseases. BioMed Res Int 2015; doi: 10.1155/2015/985814.
2. Baffour AS, Adjei J, Aryeh C et al.: Understanding the biosynthesis of platelets-derived extracellular vesicle. Immun Inflamm Dis 2015; 3: 133-140.
3. Lowry MC, Gallagher WM, O’Driscoll L: The role of exosomes in breast cancer. Clin Chem 2015, 61: 1457-1465.
4. Kriebardis A, Antonelou M, Stamoulis K, Papassideri I: Cell-derived microparticles in stored blood products: innocent-bystander or effective mediators of post-transfusion reactions? Blood Transf 2012; 10: 25-38.
5. Prudent M, Crettaz D, Delobel J et al.: Differences between calcium-stimulated and storage-induced erythrocyte-derived microvesicles. Transf Apher Sci 2015; doi.org/10.1016/j.transci.2015.10.012.
6. Salzer U, Zhu R, Luten M et al.: Vesicles generated during storage of red cells are rich in the lipid raft marker stomatin. Transfusion 2008; 48: 451-462.
7. Saas P, Angelot F, Bardiaux L et al.: Phosphatidylserine-expressing cell by-products in transfusion. A proinflammatory or anti-inflammatory effect? Transfus Clin Biol 2012; 19: 90-97.
8. Rubin O, Crettaz D, Canellini G: Microparticles in stored red blood cells: an approach using flow cytometry and proteomic tools. Vox Sang 2008; 95: 288-297.
9. Wolf P: The nature and significance of platelet products in human plasma. Br J Haematol 1967; 13: 269-288.
10. Greenwalt TJ, Steane EA, Lau FO, Sweeney-Hammond K: Aging of the human erythrocyte. [In:] Sandler SG, Nusbacher J, Schanfield MS (eds.): Immunobiology of the erythrocyte. 1st ed. Liss, New York 1980: 195-212.
11. Satta N, Toti F, Feugesas O et al.: Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J Immunol 1994; 153: 2639-2642.
12. Coombs V, Simon ACGrau GE, Arrnoux D et al.: In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. J Clin Invest 1999; 104: 93-102.
13. Minciacchi VR, Freeman MR, Di Vizio D: Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes. Semin Cell Dev Biol 2015; 40: 41-51.
14. Gould SJ, Raposo G: As we wait: coping with an imperfect nomenclature for extracellular vesicle: J Extracell Vesicles 2013; doi: 10.3402/jev.v2i0.20389.
15. Olivier G, De Jong, Bas WM et al.: Extracellular vesicles: potential roles in regenerative medicine. Front Immunol 2014; 5: 1-13.
16. Kastelowitz N, Yin H: Exosomes and microvesicles: identification and targeting by particle size and lipid chemical probes. Chembiochem 2014; 15: 923-928.
17. Frydrychowicz M, Kolecka-Bednarczyk A, Madejczyk M: Exosomes – structure, biogenesis and biological role in non-small-cell lung cancer. Scand J Immunol 2015; 81: 2-10.
18. Morel O, Jesel L, Freyssinet JM, Toti F: Cellular mechanisms underlying the formation of circulating microparticles. Asterioscler Thromb Vasc Biol 2011; 31: 15-26.
19. Maślanka K, Michur H, Smoleńska-Sym G: Mikrocząstki błon komórkowych. Acta Haematol Pol 2009; 40: 481-491.
20. Antwi-Baffour SS: Molecular characterization of plasma membrane-derived vesicles. J Biomed Sci 2015; 22: 1-7.
21. Jayachandran M, Miller VM, Heit JA, Owen WG: Methodology for isolation, identification and characterization of microvesicles in peripheral blood. J Immunol Methods 2012; 375: 207-214.
22. Xiong Z, Oriss TB, Cavaretta JP et al.: Red cell microparticle enumeration: validation of a flow cytometric approach. Vox Sanguinis 2012; 103: 42-48.
23. Orozco AF, Lewis DE: Flow cytometric analysis of circulating microparticles in plasma. Cytometry Part A 2010; 77A: 502-514.
24. Thiagarajan P, Tait JF: Collagen-induced exposure of anionic phospholipid in platelet and platelet-derived microparticles. J Biol Chem 1991; 266: 24302-24307.
25. Macey MG, Enniks N, Bevan S: Flow cytometric analysis of microparticle phenotype and their role in thrombin generation. Cytometry B Clin Cytom 2011; 80: 57-63.
26. Pattanapanyasat K, Noulsri E, Fucharoen S et al.: Flow cytometric quantitation of red blood cell vesicles in thalassemia. Cytometry Part B: Clin Cytom Part B 2004; 57 B: 23-31.
27. Lötvall J, Hill AF, Hochberg F et al.: Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 2014; doi: 10.3402/jev.v3.26913.
28. Soo CY, Song Y, Zheng Y et al.: Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells. Immunology 2012; 136: 192-197.
29. D’Souza-Schorey C, Clancy JW: Tumor-derived microvesicles: shedding light on novel microenvironment modulators and prospective cancer biomarkers. Genes Dev 2012; 26: 1287-1299.
30. Maas SLN, Vrij J: Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics. J Control Release 2015; 200: 87-96.
31. Sabatier F, Roux V, Anfosso F et al.: Interaction of endothelial microparticles with monocytic cells in vitro induces tissue factor dependent procoagulant activity. Blood 2002; 99: 3962-3970.
32. Omoto S, Nomura S, Shouzu A et al.: Detection of monocyte-derived microparticles in patients with type II diabetes mellitus. Diabetologia 2002; 45: 550-555.
33. Alkhatatbeh MJ, Enjeti AK, Acharya S et al.: The origin of circulating CD36 in type 2 diabetes. Nutr Diabetes 2013; 3: 1-7.
34. Morel O, Hugel B, Jesel L et al.: Sustained elevated amounts of circulating procoagulant membrane microparticles and soluble GPV after acute myocardial infarction in diabetes mellitus. Thromb Haemost 2004; 91: 345-353.
35. Al-Nedawi K, Meehan B, Micallef J et al.: Intercellular transfer of the oncogenic receptor EGFRv III by microvesicles derived from tumour cells. Nat Cell Biol 2008; 10: 619-624.
36. Wenche J, Ricci M, Shariatmadar S et al.: Microparticles in stored RBC as potential mediators of transfusion complications. Transfusion 2011; 51: 886-893.
37. Babaev A, Pozzi F, Hare G, Zhang H: Storage of red blood cells and transfusion-related acute lung injury. J Anesth 2014; 1: 1-6.
38. Rubin O, Canellini G, Delobel J et al.: Red blood cell microparticles: clinical relevance. Transfus Med Hemother 2012; 39: 342-347.
39. Karon BS, Hoyer JD, Stubbs JR, Thomas DD: Changes in band 3 oligomeric state precede cell membrane phospholipid loss during blood bank storage of red blood cells. Transfusion 2009; 49: 1435-1442.
40. Sparrow RL, Veale MF, Healey G, Payne KA: Red blood cell (RBC) age at collection and storage influences RBC membrane-associated carbohydrates and lectin binding. Transfusion 2007; 47: 966-968.
41. Simak J, Holada K, Risitano AM et al.: Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria. Br J Haematol 2004; 125: 804-813.
42. Sloand EM, Mainwaring L, Keyranfar K et al.: Transfer of glycosylphosphatidylinositol- anchored proteins to deficient cells after erythrocyte transfusion in paroxysmal nocturnal haemoglobinuria. Blood 2004; 104: 3782-3788.
43. Bosman GJ, Lasonder E, Groenen-Dopp YA et al.: Comparative proteomics of erythrocyte aging in vivo and in vitro. J Proteomics 2010; 73: 396-402.
44. Greenwalt TJ: The how and why of exocytic vesicles. Transfusion 2006; 46: 143-152.