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EVs in the hematopoietic microenvironment
report described that erythroleukemia cells respond to hypoxia by rapidly releasing exosomes containing miR- 486, a known regulator of erythroid differentiation, which targets Sirt1 in CD34+ HSPCs (Figure 2C).63 This confirmed and extended previous studies that had impli- cated the increased expression of miR-486-5p in support- ing erythroid differentiation of CD34+ cells in vitro.64 Conversely, the inhibition of miR-486-5p has been found to suppress CD34+ cell growth in vitro and in vivo, and decrease erythroid differentiation and survival of ery- throid cells. It is possible that a similar physiological mechanism might exist to regulate hypoxia-responsive erythropoiesis in order to increase the delivery of oxygen to starved tissues.
EVs within the BM microenvironment have been shown to modulate the behavior of HSCs in other ways. For example, treatment with pharmacological concentrations of granulocyte colony-stimulating factor used to mobilize stem and progenitor cells for collection and subsequent transplantation causes an increase in EVs containing high levels of miR-126 within the BM. These EVs are internal- ized by stroma, HSPCs and endothelial cells, delivering miR-126 into the cell, where it acts to translationally sup- press vascular cell adhesion molecule-1 (Figure 2D). This decrease in vascular cell adhesion molecule-1, along with other signaling events, results in reduced HSPC adhesion and a shift into the peripheral blood, for collection by leukapheresis.52 Experimentally, EV-contained miR-126 released from mobilized human CD34 cells conferred pro- angiogenic activity and promoted hindlimb ischemia repair.53 Another recent study found that aging and oxida- tive stress alter the miRNA content of EVs in the BM microenvironment leading to age-related stem cell dysfunc- tion. The investigators showed that BM-derived EVs from aged mice contain abundant miR-183-5p which, when endocytosed by primary BM stromal cells from young mice, decreased proliferation and inhibited osteogenic dif- ferentiation by reducing heme oxygenase 1, an enzyme essential in heme catabolism.65 Microvesicles derived from mouse embryonic stem cells were found to contain high levels of transcripts associated with pluripotency (Wnt-3 and Oct-4), and when exposed to hematopoietic progeni- tors led to their expansion.66 Additionally, hematopoietic progenitors exposed to the microvesicles derived from mouse embryonic stem cells were found to upregulate the expression of early HSC markers (SCL, HoxB4 and GATA2) and showed phosphorylation of MAPK p24/44 and serine- threonine kinase AKT.66
Finally, HSCs may contribute to their own stemness in part through secretory signaling and autocrine loops, involving vacuolar protein sorting protein 33b (VPS33B)- mediated release of exosomes as carriers of thrombopoi- etin and angiopoietin-like protein 2 and 3 (Figure 2E). Herein, the loss of VPS33B compromised HSC potential and reduced leukemogenicity in cancer models.23 This and other studies discussed in this section support the view that within the physiological BM microenvironment, HSPCs release and internalize EVs, and are broadly responsive to regulation by vesicle trafficking in order to maintain hematopoiesis.
Pathophysiological regulation of hematopoiesis by extracellular vesicles
Aside from the role of EVs in the cellular crosstalk in the BM under physiological conditions, EV trafficking also
plays a distinct role in deregulating hematopoiesis in injury and disease states, such as hematologic malignan- cies and extramedullary cancers (Table 2B).13,14,48 For exam- ple, MSC-derived EVs appear to contribute to marrow repair after radiation damage, restoring HSPC prolifera- tion and engraftment with partial restoration of peripheral blood counts after intravenous injection of MSC-derived EVs.48
We reported that acute myeloid leukemia (AML) blasts rely on EVs for the transfer of miR-150 and miR-155, which target cMyb, a highly expressed transcription factor in progenitor cells to suppress HSPC clonogenicity. The coincident downregulation of the niche retention factor CXCL12 in those studies led to HSPC mobilization into the peripheral blood (Figure 3A).13,51 These observations were extended more recently by others showing that AML EVs not only downregulate HSC-supporting factors (CXCL12, stem cell factor, and insulin-like growth factor 1) (Figure 3B), but simultaneously suppress hematopoiesis and osteolineage development by upregulating Dkk1 expression in BM stromal cells.14 On the other hand, one study showed that AML EVs increased the number of HSCs by enhancing their survival while retaining their clonogenicity and stemness with no change in the hematopoietic CD34+, CD34+CD38−, CD90+, and CD117+ phenotypes.67 Illustrating one of the key challenges in understanding HSPC regulation by EVs, neither of the two latter studies identified the specific EV component respon- sible. We and others previously showed that EVs released by steady-state or reprogrammed malignant stroma carry cytokines.17,68,69 Because most analyses of secreted cytokines do not separate vesicle-bound and vesicle-free cytokine activity it is entirely possible that some of the known cytokine activities that regulate HSPC in the leukemic niche reflect EV-mediated trafficking.
Other hematologic disorders affect hematopoiesis indi- rectly by altering the function of the supportive non- hematopoietic stroma. Both AML and myelodysplastic syndrome cells were shown to reduce the hematopoiesis- supportive capacity of MSCs by delivering miR-7977 via EVs. After uptake by MSCs, the EV-trafficked miR-7797 suppresses hematopoietic growth factors (jagged-1, stem cell factor and angiopoietin-1) by targeting the poly (rC) binding protein 1 post-transcriptional regulator (Figure 3C).70 MSCs from patients with myelodysplastic syn- drome were also shown to release EVs that traffic miR- 10a and miR-15a to CD34+ progenitor cells, causing the transcriptional regulation of MDM2 and P53 genes, alter- ing HSPC viability and clonogenicity.71 EVs released from chronic myelogenous leukemia cells have also been impli- cated in altering the BM microenvironment by activating epithelial growth factor receptor signaling in stromal cells. Chronic myelogenous leukemia exosomes were shown to contain amphiregulin, an epithelial growth factor recep- tor-activating ligand that leads to the downstream expres- sion of matrix metalloproteinase-9 and interleukin-8, giv- ing leukemic cells an adhesive and proliferative advantage within the hematopoietic niche.72
Extramedullary cancers, such as melanoma, also use EVs for the endocrine regulation of BM progenitors. For exam- ple, one study showed that melanoma EVs mobilize BM progenitors by targeting the receptor tyrosine kinase, c- MET, in turn upregulating pro-inflammatory molecules at sites of macrophage trafficking to promote their invasion and metastasis in distant organs.73
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