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JAK2V617F endothelial cells are pro-thrombotic
Discussion
Despite significant advances in deciphering the molec- ular mechanisms responsible for the occurrence and transformation of MPN, the mechanisms that lead to thrombosis, the main cause of morbidity and mortality, remain largely elusive. Recent identification of the JAK2V617F mutation in EC of patients with MPN13-15 opened new perspectives in the pathogenesis of thrombosis in MPN. Here we demonstrate that JAK2V617F-positive EC promote spontaneous thrombosis under basal conditions and have an increased thrombotic response to weak inflammatory stimuli. We describe that the mechanism that leads to thrombosis involves endothelial P-selectin release and cell surface exposure and subsequent leuko- cyte rolling and adhesion. We also describe that treat- ment with hydroxy-urea decreases P-selectin endothelial expression and thrombus formation in mice expressing JAK2V617F only in EC.
We used a mouse model that allows expression of JAK2V617F only in EC to ensure that the phenotype observed in these mice was solely due to the presence of JAK2V617F EC. We are aware that there are differences between our model and patients with MPN, who often have JAK2V617F - positive blood cells together with some JAK2V617F EC. Moreover, not all patients have JAK2V617F EC and, for those that do, it is currently not known where they are located. Our mouse model is not, therefore, representative of the human situation and is not meant to be a model of MPN; however, it does allow precise characterization of the properties of JAK2V617F EC.
The link between P-selectin expression and thrombosis has been described previously, and in most cases P- selectin originates from platelets.28,31,32 The mechanism of P-selectin-mediated thrombosis involves neutrophil acti- vation, either through tissue factor expression and activa- tion of the extrinsic coagulation pathway,28 or through priming for neutrophil extracellular trap formation,33 a process that is now well-recognized to participate in thrombus formation.34 An increase in endothelial P-selectin and subsequent thrombosis were seen in response to venous flow reduction and local hypoxia.28 Here, we show for the first time that EC can have consti- tutively increased expression of P-selectin, even without any hypoxic or inflammatory stimuli. Further studies are now required to decipher the specific intracellular mecha- nism responsible for increased P-selectin expression in JAK2V617F-expressing EC. One possible mechanism could be increased STAT3 phosphorylation, as STAT3 activation has been shown to upregulate expression of P-selectin in EC35 (Online Supplementary Figure S7).
Given that MPN are acquired hematologic malignan- cies, the description of pro-thrombotic JAK2V617F EC raises the question of their origin. JAK2V617F EC have been found in microdissected vessels13,14 and after culture of circulat- ing endothelial progenitors, both in colony-forming unit - endothelial cell (CFU-EC)15,36-38 and endothelial colony- forming cells (E-CFC).15,38 The finding that JAK2V617F CFU- EC are present in all MPN patients was not surprising given that CFU-EC are of hematopoietic origin. However, E-CFC are not of hematopoietic origin and the presence of JAK2V617F EC thus suggests the existence of a progenitor cell of hematopoietic and endothelial lineages. Such cells certainly exist in the embryo but their existence in adults is a matter of debate. In the case of microdissection exper-
iments,13,14 it is possible that the JAK2V617F EC that have been microdissected derive from CFU-EC.39 The presence of JAK2V617F EC of real endothelial origin in MPN is prob- ably rare, but the presence of JAK2V617F EC of hematopoi- etic origin is common. Demonstrating that JAK2V617F EC have a pro-thrombotic phenotype is thus particularly rel- evant to our understanding of the pathogenesis of throm- bosis in MPN.
Our study has important therapeutic implications. We demonstrate that treatment with hydroxyurea inhibits the pathological hyper-adhesive phenotype of JAK2V617F EC. The results presented here challenge current thinking, according to which the anti-thrombotic effect of hydroxy-urea in MPN is only mediated by lowering the blood cell count. However, a direct effect of hydroxyurea on EC has been reported40,41 and is thought to occur via stimulation of the nitric oxide-cyclic GMP pathway in EC, and reduction of leukocyte rolling.30 Moreover, in patients with sickle cell disease, treatment with hydrox- yurea efficiently decreases the frequency of vaso-occlu- sive crises.42 These findings suggest that hydroxyurea should be considered for patients with MPN and a history of thrombosis. This is often the case, as hydroxyurea is the first-line therapy in high-risk patients. However, in young patients, hydroxy-urea is usually avoided because of its potential leukemic effect, even if large, retrospective studies have not confirmed this effect.43-45 The antithrom- botic and leukemic effects of hydroxyurea should thus be addressed in a prospective cohort of young MPN patients with thrombosis.
Another therapeutic implication of our work comes from the demonstration that increased endothelial P-selectin favors thrombosis in MPN, as was shown in sickle cell disease.46 Very recently, a clinical trial demon- strated that treatment with an anti-P-selectin antibody, crizanlizumab, efficiently prevented pain crises in patients with sickle cell disease.47 It is thus tempting to speculate that such treatment could have therapeutic benefits in MPN patients at high risk of thrombosis, who need to receive anticoagulant treatment for a MPN-related throm- bosis (such as splanchnic thrombosis).
Our description of the pro-thrombotic profile of JAK2V617F EC may appear to contradict with the results of Etheridge et al.48 These authors used Tie2-Cre/FF1 mice, which allow constitutive expression of human JAK2V617F in blood and EC. These mice were irradiated then transplant- ed with the bone marrow of wild-type mice to generate mice in which only the EC express JAK2V617F. Etheridge et al. reported a hemorrhagic diathesis due to acquired von Willebrand disease. There are several possible reasons for the apparently contradictory results. Studies have shown the importance of the JAK2V617F :JAK2WT ratio for the MPN phenotype49 and Etheridge et al. used transplanted mice with a human JAK2V617F :mouse JAK2WT ratio of 1:5 in EC, whereas we used a tamoxifen-inducible mouse model that allows heterozygous expression of mouse JAK2V617F/JAK2WT specifically in EC. Another major differ- ence lies in the models of thrombosis, as Etheridge et al. used models in which the endothelial layer is disrupted whereas we chose three models in which the endothelial barrier is maintained intact. Of note, Etheridge et al. reported an overall increase in the content of vWF in EC, which is in accordance with our results.
Altogether, we believe that our work is clinically rele- vant for three reasons; (i) it suggests that specific biomark-
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