Moving beyond size and phosphatidylserine exposure: evidence for a diversity of apoptotic cell‐derived extracellular vesicles in vitro

Abstract

Apoptosis is a form of programmed cell death that occurs throughout life as part of normal development as well as pathologic processes including chronic inflammation and infection. Although the death of a cell is often considered as the only biological outcome of a cell committed to apoptosis, it is becoming increasingly clear that the dying cell can actively communicate with other cells via soluble factors as well as membrane-bound extracellular vesicles (EVs) to regulate processes including cell clearance, immunity and tissue repair. Compared to EVs generated from viable cells such as exosomes and microvesicles, apoptotic cell-derived EVs (ApoEVs) are less well defined and the basic criteria for ApoEV characterization have not been established in the field. In this study, we will examine the current understanding of ApoEVs, in particular, the ApoEV subtype called apoptotic bodies (ApoBDs). We described that a subset of ApoBDs can be larger than 5 μm and smaller than 1 μm based on flow cytometry and live time-lapse microscopy analysis, respectively. We also described that a subset of ApoBDs can expose a relatively low level of phosphatidylserine on its surface based on annexin A5 staining. Furthermore, we characterized the presence of caspase-cleaved proteins (in particular plasma membrane-associated or cytoplasmic proteins) in samples enriched in ApoBDs. Lastly, using a combination of biochemical-, live imaging- and flow cytometry-based approaches, we characterized the progressive lysis of ApoBDs. Taken together, these results extended our understanding of ApoBDs.

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

“…However, phosphatidylserine is also exposed on apoptotic bodies, which are larger vesicles specifically formed during the late stages of apoptosis [97,98]. Apoptotic bodies can be distinguished from other phosphatidylserine-positive MVs based on positivity for caspases 3 and 7 and their substrates (e.g., ROCK1 and PANX1) [99], while size is unreliable, because apoptotic bodies (~0.1 to~5 µm) overlap in size with other EV subtypes [99]. Here, it should be remarked that phosphatidylserine exposure, although often used as a surrogate marker of apoptosis [100], does not distinguish apoptotic bodies from other MVs [84,99].…”

Extracellular Vesicles in Feto–Maternal Crosstalk and Pregnancy Disorders

“…Phosphatidylserine is also expressed by apoptotic body membranes, and for this reason, the positivity to Annexin V (which is related to the surface exposure of phosphatidylserine) is not a useful method that allows to distinguish apoptotic bodies and microvesicles derived from activated cells. Apoptotic bodies must be identified, instead, for their positivity to the caspase substrates [33].…”

“…Apoptotic bodies are membrane vesicles of approximately 50-2000 nm in diameter, released during the programmed cell apoptosis processes ( Figure 1) [30,34]. Given that apoptotic cells express phosphatidylserine on their surface [35], the positivity to Annexin V (which binds phosphatidylserine) has been considered a hallmark of apoptotic bodies, even if the enrichment in caspase 3 and 7 has been proposed to be more specific for apoptotic bodies [33]. It has been recently demonstrated that, as with all the other EV subtypes, apoptotic bodies represent an active vehicle of intercellular communication from dying to living cells [14].…”

Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking

“…Finally, the release of distinct ApoBDs from the cell occurs [ 60 ]. As with exosomes and MVs, ApoBDs are enriched in proteins associated with their biogenesis, including caspase 3-cleaved ROCK1, PlexB2 and PANX1 [ 61 , 62 ]. In addition, cytokines, growth factors and nuclear material have also been detected within ApoBDs [ 24 , 47 , 63 ].…”

“…Whether these EVs were generated through energy-dependent mechanisms, such as exosome formation or membrane shedding, or their specific cargo, was not determined. Furthermore, as it has been previously shown the ApoBDs can lyse over time [ 62 ]; the ability of ApoBDs undergoing lysis to subsequently reform into more EVs is also of interest.…”