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JAK2V617F endothelial cells are pro-thrombotic
completely abrogated thrombus formation in Pdgfb- iCreERT2;JAK2V617F/WT mice, but had no effect in control mice (Figure 6E). These results demonstrate that the pro- thrombotic phenotype of JAK2V617F EC is primarily the con- sequence of increased adhesive properties, due to overex- pression of membrane P-selectin.
Treatment with hydroxyurea abrogates tumor necrosis factor-induced thrombosis in Pdgfb-iCreERT2;JAK2V617F/WT mice through decreased P-selectin expression
Hydroxyurea is an anti-metabolite frequently used in MPN to reduce the occurrence of thrombosis. Its anti- thrombotic effect is reported to occur via the reduction of blood cell counts. Hydroxyurea is also used in sickle cell disease to reduce vaso-occlusive crises, and its beneficial effect is in part mediated by a direct effect on EC, with a
reduction in leukocyte adhesion.30 We hypothesized that hydroxyurea might be capable of reducing the pro-throm- botic effect of JAK2V617F EC. To test this, we treated Pdgfb- iCreERT2;JAK2V617F/WT and control mice with hydroxyurea for 10 days, injected a low dose of TNF-α and quantified thrombus formation in the lungs after 4 h. We observed a significant inhibition of thrombus formation in Pdgfb-iCreERT2;JAK2V617F/WT mice (Figure 7A). We then investigated whether hydroxyurea had modified the adhesiveness of the EC. We observed a reduction in leuko- cyte rolling and adhesion in mesenteric venules 4 h after TNF-α administration in Pdgfb-iCreERT2;JAK2V617F/WT mice treated with hydroxyurea (Figure 7B,C). To further deter- mine whether this was due to a direct effect on EC, we treated JAK2V617F HUVEC with 100 mM hydroxyurea for 24 h, washed them and analyzed neutrophil adhesion in vitro. Interestingly, there was a significant reduction of neu-
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D
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Figure 6. Increased endothelial P-selectin expression is responsi- ble for the pro-adhesive phenotype of JAK2V617F-expressing endothelial cells. (A,B) Under static conditions, increased adhesion of (A) normal mononuclear cells and (B) neutrophils on JAK2V617F human umbilical vein endothelial cells is reversed in the presence of a P-selectin blocking antibody (Ab). Results are mean value ± SEM from three experiments. In Pdgfb-iCreERT2;JAK2V617F/WT mice, increased (C) rolling and (D) adhesion of leukocytes is abolished in the presence of a P-selectin blocking antibody. (E) Increased thrombus formation in Pdgfb-iCreERT2;JAK2V617F/WT mice is abrogat- ed in the presence of a P-selectin blocking antibody. Results are expressed as mean value ± SEM. Statistical significance was deter- mined by two-way analysis of variance and a Sidak post-hoc test. *P<0.05; **P<0.01; ****P<0.0001.
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