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EVs in the hematopoietic microenvironment
of neutrophils that consistently shed two distinct narrowly defined vesicle populations of ~100 nm and ~500 nm, both budding at the limiting membrane.28
Large vesicles
Large vesicles, also referred to as large oncosomes due to their tumor-derived origin, are a class of EVs that can reach up to 10 microns in size and contain intact organelles and an ordered cytoskeletal structure.29 Large vesicles are similar to apoptotic bodies in size and compo- sition; however, unlike apoptotic bodies, large vesicles are formed from cleavage of cytoplasmic extensions from intact living cells. Large vesicles have been described in B- cell acute lymphoblastic leukemia and prostate cancer, and have been demonstrated within patients’ samples and in vitro cultures of cancer cell lines.29,30
Apoptotic bodies
Apoptotic bodies emerge during the course of pro- grammed cell-death, as nuclear karyorrhexis occurs with cytoplasm and surrounding plasma membrane beginning to bleb into fragments.31 Apoptotic bodies consist of an intact plasma membrane enclosing cytosolic components and can contain both organelles and nuclear fragments. These bodies are subsequently eliminated through phago- cytosis by surrounding cells and degraded in phagolyso- somes.31 It has been reported that apoptotic bodies can hor- izontally transfer DNA to phagocytic recipient cells. As an example of this, one study showed that Epstein-Barr virus- infected B-lymphocytes generate apoptotic bodies that carry viral DNA and aid in the transfer of the virus to unin- fected cells.32
Vesicle fate
Once released from the parent cell, EVs can follow mul- tiple routes. Some cancer cells generate EVs that rupture soon after release from their parent cells, distributing enzymes such as vascular endothelial growth factor and matrix metalloproteases into the surrounding interstitial space in order to promote angiogenesis and support cancer invasion through metastatic dissemination.33,34 EVs released into the blood appear to have a short half-life in circulation. In one representative study of B16-BL6 melanoma-derived EVs packaged with luciferase and lactadherin, luciferase activity was lost within minutes of intravenous injection with an observed serum half-life of approximately 2 min- utes followed by rapid redistribution into tissues.35
A broad range of mechanisms for cellular uptake have been identified for EVs, including membrane fusion, phagocytosis or receptor-mediated caveolin-, clathrin- or lipid raft-mediated endocytosis, all culminating with trans- port of the EV cargo directly into the intracellular compart- ment.36 The differences from study to study suggest that EV uptake is a variable process and likely dependent on the type of EV and the parent and recipient cells involved. Experiments have shown that uptake is prevented at lower temperatures, suggesting that internalization is energy dependent and does not occur as a passive process.37 The uptake of EVs can be partially blocked by treating vesicles with either heparan sulfate or proteinase K, indicating a role for proteoglycans and surface proteins, respectively, in gaining entry into the cell.37,38 Pre-treatment of cells with the actin-depolymerizing drug cytochalasin D prior to EV exposure prevents cytoskeletal remodeling and reduces EV internalization,39 The use of the dynamin 2 inhibitor dyna-
sore, which abrogates caveolin/clathrin-mediated endocy- tosis, has also been shown to inhibit uptake of reticulo- cyte-derived exosomes by macrophages.40 These data taken together are suggestive of an endocytic process mediating vesicle internalization. Little is known about specific mechanisms of uptake within the hematopoietic niche, although one study reported that megakaryocyte- derived EVs gain entry into hematopoietic progenitors cells via lipid raft mediated endocytosis, macropinocytosis and membrane fusion.61 Further study is warranted in order to understand the cellular events by which HSPC and sup- portive cells of the bone marrow differentially regulate the process of EV entry.
How EVs are specifically targeted to different cell types within the hematopoietic niche in order to regulate hematopoiesis remains largely unknown. Among the most abundant membrane-associated proteins found on EVs are tetraspanins, a large cell-surface protein superfamily that interacts with transmembrane proteins and cytosolic sig- naling molecules to facilitate the organization of these structures into microdomains.41 Tetraspanins have been linked to many functions: intracellular signaling through G- protein coupled receptors and protein kinase C; migration and metastasis by interacting with integrins and vascular cell adhesion molecule; cell morphogenesis by direct bind- ing of alpha-actinin and the induction of actin polymeriza- tion.42,43 EV-embedded tetraspanins are dependent on the type of their parent cell; however, CD9, CD63, CD81, CD82, and CD151 are enriched in EVs derived from a range of sources.22 CD9, a common tetraspanin used to identify EVs was previously described in association with c-kit/CD117, a tyrosine kinase receptor that is highly expressed on HSPCs.44 Tetraspanins such as CD37, CD53 and TSSC6 have been found exclusively on hematopoietic cells. It is known that these tetraspanins interact with hematopoietic-specific targets such as Src homology region 2 domain-containing phosphatase-1, the pattern recognition receptor dectin-1, MHC-I/II, integrin α4β1, T- cell/NK-cell co-stimulatory CD2, as well as common signal transducers including phosphatidylinositol-3 kinase and protein kinase C.45 Hematopoietic-specific tetraspanins and integrins on the EV surface remain strong candidates in tar- geting vesicles to specific cell types within the hematopoi- etic niche.
A recent study demonstrated that, once inside the target cell, EVs are sorted into the endosomal pathway, move quickly through the cytoplasm and then stall at the endo- plasmic reticulum, before eventually fusing with lyso- somes for degradation.46 The process of cargo release by internalized EVs remains to be clarified. As the principal compartment for translation within the cell, the endoplas- mic reticulum is a likely site for the deposition of mRNA and miRNA cargo. This and the assembly of the RNA interference-silencing complex in the endoplasmic reticu- lum may potentially explain how EVs alter protein synthe- sis and change cellular behavior. The half-life of internal- ized EVs has not been well defined. In the same study, 293T-derived EVs remained intact for hours to days once inside primary fibroblasts, with 50-60% merging with lysosomes by 48 hours.46
Physiological regulation of hematopoiesis by extracellular vesicles
The BM comprises hematopoietic and non-hematopoi- etic cells organized into specialized microenvironments
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