MINI REVIEW ARTICLE Front. Physiol., 05 May 2017
Carina Hromada1,2, Severin Mühleder1,2, Johannes Grillari2,3,4, Heinz Redl1,2 and Wolfgang Holnthoner1, 2*
1AUVA Research Centre, Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria, 2Austrian Cluster for Tissue Regeneration, Vienna, Austria, 3Christian Doppler Laboratory on Biotechnology of Skin Aging, Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria, 4Evercyte GmbH, Vienna, Austria
Extracellular vesicles, including exosomes, microparticles, and apoptotic bodies, are phospholipid bilayer-enclosed vesicles that have once been considered as cell debris lacking biological functions. However, they have recently gained immense interest in the scientific community due to their role in intercellular communication, immunity, tissue regeneration as well as in the onset, and progression of various pathologic conditions. Extracellular vesicles of endothelial origin have been found to play a versatile role in the human body, since they are on the one hand known to contribute to cardiovascular diseases, but on the other hand have also been reported to promote endothelial cell survival. Hence, endothelial extracellular vesicles hold promising therapeutic potential to be used as a new tool to detect as well as treat a great number of diseases. This calls for clinically approved, standardized, and efficient isolation and characterization protocols to harvest and purify endothelial extracellular vesicles. However, such methods and techniques to fulfill stringent requirements for clinical trials have yet to be developed or are not harmonized internationally. In this review, recent advances and challenges in the field of endothelial extracellular vesicle research are discussed and current problems and limitations regarding isolation and characterization are pointed out.
Keywords: extracellular vesicles, endothelial cells, exosomes, microparticles, pathology
Extracellular vesicles (EVs) are a heterogeneous population of phospholipid bilayer-enclosed vesicles that are secreted into the extracellular space by several cell types (Yáñez-Mó et al., 2015). Although once considered as cell debris lacking biological functions, EVs have recently become a focal point of interest in research with respect to their importance in the regulation of immune responses, contribution to the onset and progression of diverse pathologies such as age-associated diseases like neurodegenerative and cardiovascular diseases (CVDs), as well as their therapeutic potential (El Andaloussi et al., 2013; Weilner et al., 2013). EVs are commonly classified into three major subtypes based on vesicle biogenesis as well as size: exosomes, microparticles (MPs) or microvesicles, and apoptotic bodies. Ranging from approximately 30–100 nm in size, exosomes represent the smallest population among EVs. They are formed as intraluminal vesicles inside multivesicluar bodies (MVBs) in the endosomal compartment during the maturation of early into late endosomes (van der Pol et al., 2012; Weilner et al., 2013; Colombo et al., 2014). These MVBs subsequently either fuse with lysosomes to be degraded, or with the plasma membrane to be released as exosomes. The formation of MVBs is mostly mediated by the endosomal sorting complex required for transport (ESCRT) machinery, which consists of four complexes comprising approximately 30 proteins that overall sequester ubiquitinated transmembrane proteins in the endosomal membrane, and promote bud formation with sorted cargo and subsequent scission. However, MVB formation might also occur in an ESCRT-independent manner, e.g., via the tetraspanin CD63, the lipid metabolism enzymes sphingomyelinase, and phospholipase D2. Moreover, SNARE and Rab proteins (RAB7, RAB11, RAB27, and RAB35) seem to be involved in exosome secretion (Colombo et al., 2014). MPs, on the other hand, range between 100 and 1,000 nm and emerge directly from the outward budding and fission of the cell membrane (Combes et al., 1999; Heijnen et al., 1999; György et al., 2011; van der Pol et al., 2012). The formation of outward buds is driven by several membrane rearrangements due to increased Ca2+ levels: the enzymes flippase, floppase, and scramblase are recruited and activated to modify the lipid composition of the plasma membrane (i.e., the externalization of phosphatidylserine (PS), one major feature of MPs), and the protein calpain is furthermore activated to cleave cytoskeletal proteins to remodel the cytoskeleton. Additionally, also ARF6 and components of the ESCRT family have been implicated in the formation and release of MPs (Colombo et al., 2014; Minciacchi et al., 2015). The largest extracellular vesicles are apoptotic bodies released from dying cells and range from 1 to 5 μm in diameter (György et al., 2011; van der Pol et al., 2012).
The composition of EVs seems to be strongly influenced by the type and (patho) physivological condition of the secreting cell, the stimuli triggering their release, and the different pathways of EV biogenesis. Exosomes carry lipids, miRNAs, mRNAs, and proteins such as tetraspanins (CD9, CD63, and CD81), integrins, heat shock proteins (Hsp60, Hsp70, and Hsp90), ESCRT proteins (TSG101 and Alix), annexins, Rab proteins, GTPases, and flotillin (Mathivanan et al., 2010; van der Pol et al., 2012; Kourembanas, 2015). MPs also carry lipids (PS, cholesterol) and proteins including integrins, selectins, CD40L and MHC I and II (Safdar et al., 2016). Despite seemingly strong variations in size and features, there is still a demand to identify markers for distinguishing certain extracellular vesicle subpopulations in order to be able to truly understand the molecular mechanisms of biogenesis, secretion, and uptake as well as to assess the biological functions of the respective subtypes. In fact, there are several overlapping properties of exosomes and MPs that have led to the suggestion to collectively refer to them as “extracellular vesicles”: (i) size ranges cannot be considered absolute, (ii) lack of specific markers to uniquely identify a certain subtype, (iii) simultaneous release of all the different subtypes of EVs, and (iv) the impossibility to exclusively isolate pure fractions of a certain vesicle subtype from biological fluids or conditioned cell culture media (György et al., 2011; Gould and Raposo, 2013; Witwer et al., 2013). Therefore, the aim of this review is to discuss current problems regarding the isolation and characterization of EVs and summarize the versatile roles of endothelial extracellular vesicles in the human body as well as stimuli that trigger their release.