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CD150+CD48– HSPCs expressing phosphorylated EGFR was increased in irradiated JAK2V617F HSPCs (from Tie2/FF1 mice) as compared to irradiated JAK2WT HSPCs (from control mice) (2.7-fold; P=0.042), suggesting that EGFR signaling activity was up-regulated in irradiated JAK2V617F HSPCs (Figure 4D). These results suggest that the JAK2V617F-bearing vascular niche contributes directly to HSPC radioprotection, possibly by its elaboration of soluble niche factors.
Discussion
Vascular ECs are a major component of the HSPC niche (the “vascular niche”) and provide many key factors that are required for HSPC maintenance.9 Patients with MPNs are characterized by increased marrow angiogenesis com- pared to normal marrow.10-12 ECs carrying the JAK2V617F mutation can be detected in patients with MPNs, suggest- ing that ECs are involved in the pathogenesis of MPNs.15,18 Here, by using the hematopoietic and endothelial specific Tie2-Cre system and different marrow transplantation models, we have been able to highlight the importance of JAK2V617F-bearing ECs in MPN disease relapse, which is seen in up to 40% of patients (especially after reduced intensity conditioning) following allogeneic SCT, the only curative treatment for MPNs.6,8,42-44
It has long been known that hematopoietic recovery fol- lowing lethal irradiation requires an intact vasculature.29-34 Following radiation injury, co-culture of irradiated HSPCs with ECs can rescue HSPCs with multilineage reconstitut- ing capacity.45,46 Our previous study has demonstrated that JAK2V617F-bearing ECs proliferate to a greater extent than JAK2WT ECs in vitro.21 In this study, we show that the JAK2V617F-bearing ECs display less cell apoptosis in vitro after irradiation compared to JAK2WT ECs. In addition, the JAK2V617F-mutant Lin– HSPCs produce more cells and hematopoietic colonies after irradiation when cul- tured on JAK2V617F-bearing ECs compared to their being cultured on JAK2WT ECs. Moreover, the expression levels of CXCL12, EGF, and PTN, which are important niche fac- tors involved in HSPC maintenance and/or HSPC regener- ation following radiation injury, were up-regulated in irra- diated JAK2V617F-bearing ECs compared to JAK2WT ECs (Figures 3 and 4). These results suggest that the JAK2V617F-bearing vascular niche contribute directly to HSPC radioprotection. Consistent with these findings, 7 of 12 mice in our Tie2/FF1 recipients of normal marrow demonstrated mixed chimerism of an average 77% donor in peripheral blood cells at fourteen weeks following transplantation (Figure 1B). Previously we reported that the JAK2V617F-bearing vascular niche promotes the expansion of the JAK2V617F HSPCs in preference to JAK2WT HSPCs and the development of marrow fibro- sis.20 Since graft failure or poor graft function in MPN patients after SCT is most likely due to marrow fibrosis,44 our work has demonstrated that the mutant vascular niche can contribute to the poor donor cell engraftment and the high incidence of disease relapse, the two major causes of treatment-related morbidity and mortality asso- ciated with allogeneic SCT in patients with MPNs.2,6,7,44
The Tie2-Cre mice express Cre recombinase in both ECs and hematopoietic cells. Although an EC-specific Cre (e.g. VEcadherin-Cre) would allow us to distinguish the specific role of ECs in HSPC radioprotection, we chose to
use Tie2-Cre as it mimics the human MPNs in which both the HSPCs and ECs harbor the JAK2V617F mutation. In order to determine whether the radioprotection pheno- type we have observed in the Tie2/FF1 mice is also due to the JAK2V617F mutation in HSPCs, we generated a chimeric murine model with JAK2V617F-mutant HSPCs and a WT vascular niche using marrow transplantation. We found that, in the WT vascular niche, the JAK2V617F- mutant Lin- HSPC is more (not less) sensitive to radiation- induced apoptosis than JAK2WT HSPCs. Although there have been reports that EC infusion could augment hematopoietic recovery following myeloablative injury, transplanted ECs exert their pro-regenerative effect tran- siently, and there is no evidence that donor marrow ECs could engraft and achieve long-term reconstitution in the recipient marrow vascular niche.31,32,47 Therefore, analysis six weeks post transplantation (Figure 2A and B) is unlike- ly to be affected by “carry-over” ECs from the Tie2/FF1 donor at the time of transplantation. In addition, the engraftment potential of irradiated JAK2V617F-mutant HSPCs does not differ from irradiated JAK2WT HSPCs (Figure 2). The results of these studies suggest that the radioprotection phenotype we have observed in the Tie2/FF1 recipients is unlikely to be due solely to the pres- ence of the JAK2V617F mutation in HSPCs. In contrast, in another chimeric murine model with WT HSPCs and JAK2V617F-bearing vascular niche, WT Lin– HSPC is less sensitive to radiation-induced apoptosis in the JAK2V617F-mutant vascular niche compared to WT vas- cular niche (Figure 3). These results suggest that JAK2V617F-bearing vascular niche contributes directly to HSPC radioprotection.
We could not exclude the possibility that altered inter- actions between the JAK2V617F HSPCs and JAK2V617F ECs contribute to HSPC radioprotection in the Tie2/FF1 mice. Indeed, no significant difference was observed in cell numbers or hematopoietic progenitors between JAK2WT HSPCs cultured on JAK2V617F-bearing ECs compared to their being cultured on JAK2WT ECs after irradiation (Figure 3A and B). This observation suggests that the JAK2V617F-bearing vascular niche by itself may not be sufficient to account for the radioprotection pheno- type. Rather, it is most likely that specific cell-cell interac- tions involving the stem cells and niche ECs are required to provide the radioprotection of JAK2V617F HSPCs when present in a JAK2V617F vascular niche, as exempli- fied by the up-regulated EGF-EGFR signaling reported in this study (Figure 4). Systemic analysis of HSPC and EC proteins using either quantitative proteomics or antibody- based arrays, along with specific knock-out mouse models would be required to further investigate the interactions between HSPCs and ECs in JAK2V617F-bearing MPNs in vitro and in vivo.
Although the JAK2V617F mutation has only been reported in liver and spleen ECs from patients with MPNs,15,18 it is very probably also present in their marrow ECs, considering that liver, spleen, and marrow are all hematopoietic organs during embryonic development and/or throughout adulthood. In most MPN patients, the stem cell compartment in MPN is heterogeneous with the presence of both JAK2 wild-type clones and the JAK2V617F mutant clones. We hypothesize that the vas- cular niche in MPN patients is also heterogeneous with the co-existence of both normal and mutant ECs. Since the JAK2V617F mutation is present in all HSPCs and ECs
haematologica | 2018; 103(7)