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A. Gao et al.
addition, apoptosis-associated genes were significantly negatively enriched in MK from AML BM (Figure 3D). These data indicated that the maturation of MK was seri- ously impaired in AML BM.
The maturation of MK and their platelet-releasing activity have been proven to be dependent on their inter- action with vascular endothelium.36 We wondered if the defective maturation of MK in AML BM was caused by disrupted interaction with BM endothelial cells. As expected, a variety of genes involved in leukocyte migra- tion, cell adhesion, cytokine-receptor interactions and chemokine signaling pathways were downregulated in MK from AML BM versus control BM (Figure 3E, F), indi- cating their weakened crosstalk with endothelial cells and the extracellular matrix. We then determined the location of MK with respect to blood vessels by in situ immunoflu- orescence imaging. In AML BM, we observed severe destruction of normal vasculature, including lack of typi- cal sinusoid structure and vascular lumens (Figure 3G). Meanwhile, MK in AML BM were located closer to BM endothelial cells (Figure 3G, H), as a result of solid stress
applied to them by overgrowing leukemia blasts. The compression of blood vessels and impaired blood perfu- sion in these areas might reduce the contribution of adja- cent MK to the platelet pool.
Interleukin-4 signaling was upregulated in acute myeloid leukemia bone marrow and exerted inhibitory effects on multiple stages of megakaryocyte differentiation
As thrombopoietin is a key regulator of MK, we exam- ined its concentration in the serum of control and AML mice. Thrombopoietin levels were similar in the two groups (Online Supplementary Figure S5), indicating that thrombopoietin is not the main cause of thrombocytope- nia in our AML mouse model. To determine the factors in the leukemia microenvironment that had a negative impact on MK differentiation and maturation, we referred to our cytokine array data of AML versus control BM plasma.12 Six cytokines (CCL3, CCL27, IL-4, Tnfrsf1a, Tnfrsf1b and Fcgr1) were upregulated in AML BM plas- ma. Among them, IL-4 has been reported to inhibit
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haematologica | 2019; 104(10)
Figure 2. Reduced megakaryocyte differentiation of long-term hematopoietic stem cells and MPP2 from acute myeloid leukemia bone marrow. (A) Representative flow cytometric plots of vWF+ cells in LT-HSC and MPP2 from healthy control and acute myeloid leukemia (AML) bone marrow (day 14). (B) Percentage of vWF+ cells in LT-HSC and MPP2 from healthy control and AML bone marrow. Five to six mice, three independent experiments. (C, D) Percentage of donor-derived eGFP+ platelets (C) and nucleated cells (D) in peripheral blood on day 14 after transplantation of 400 LT-HSC and 250 MPP2 isolated from control or AML bone marrow (day 14). Six mice, two independent experiments. *P<0.05, **P<0.01, ***P<0.001. ns, no significant difference. Error bars represent the standard error of mean. vWF: von Willebrand factor; LT-HSC: long-term hematopoietic stem cells; Ctrl: control; MPP: multipotent progenitor; eGFP: enhanced green fluorescent protein.