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JAK2V617F-bearing vascular niche in MPNs
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Figure 4. The JAK2V617F mutation alters vascular niche function to con- tribute to hematopoietic stem/progenitor cell (HSPC) radioprotection. (A) After 300cGy irradiation, cell apoptosis was higher in the JAK2WT endothelial cells (ECs) than JAK2V617F ECs. Data are from 1 of 3 inde- pendent experiments that gave similar results. (B and C) The expression levels of CXCL12, EGF and PTN in unirradiated (B) and irradiated (C) JAK2V617F-bearing ECs compared to irradiated JAK2WT ECs. Gene expression is shown as the relative fold-change compared with the JAK2WT EC expression which was set as 1. (D) Representative histogram plots and flow cytometry quantitative analysis of phosphorylated EGFR expression in irradiated JAK2WT HSPCs [from wild-type (WT) control mice] and JAK2V617F HSPCs (from Tie2/FF1 mice) (n=2 in each group).
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angiogenesis in vitro compared to JAK2WT ECs. In addi- tion, the tubular structures formed by the JAK2V617F- bearing ECs in vitro were more stable than those from JAK2WT ECs.21 In this study, we found that JAK2V617F lung ECs displayed less cell apoptosis in vitro after 300cGy irradiation compared to JAK2WT ECs (7.7% vs. 19.5%; P=0.026) (Figure 4A). It has long been known that hematopoietic recovery following lethal irradiation requires an intact vasculature.29-34 Therefore, the increased cell proliferation and/or decreased apoptosis could expand the vascular niche in JAK2V617F-bearing mice, which in turn contributes to the hematopoietic radioprotection we have observed in the Tie2/FF1 recipient mice.
CXCL12 is an essential niche factor important for both HSPC maintenance and HSPC survival after radiation injury.35-38 Epidermal growth factor (EGF) and pleiotrophin (PTN), two other factors secreted by the vascular niche,
have been shown to play important roles in the regulation of HSPC regeneration following radiation injury.39-41 Recently, we demonstrated that the expression level of CXCL12 was up-regulated in JAK2V617F-bearing marrow ECs compared to wild-type ECs, which could mediate the clonal expansion of JAK2V617F HSPCs, via the up-regulat- ed CXCL12 receptor CXCR4, over JAK2WT HSPCs.20 To understand the EC signals responsible for HSPC radiopro- tection in the Tie2/FF1 recipient mice, we measured the expression levels of CXCL12, EGF, and PTN in both non- irradiated and irradiated JAK2WT and JAK2V617F lung ECs. qPCR analysis confirmed that there was upregulation of CXCL12 (2.5-fold; P=0.0001), EGF (4.0-fold; P=0.011) and PTN (11.4-fold; P=0.00001) in irradiated JAK2V617F- bearing ECs compared to irradiated JAK2WT ECs (Figure 4B and C). Furthermore, quantitative flow cytometry analysis showed that the proportion of marrow
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