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A. Guy et al.
JAK2V617F endothelial cells have normal anticoagulant activity
Next, we sought to decipher the mechanisms by which JAK2V617F expression in EC leads to a pro-thrombotic phe- notype. To assess whether the expression of JAK2V617F by EC decreased these cells’ anticoagulant properties or even triggered pro-coagulant properties, we measured throm- bin generation at the surface of HUVEC transduced with lentivirus encoding for either human JAK2V617F or wildtype JAK2 (JAK2WT), or with an empty lentivirus as a control. Our data show that HUVEC were efficiently transduced
as: (i) 95% of JAK2V617F or JAK2WT HUVEC were GFP-posi- tive as assessed by flow cytometry (Online Supplementary Figure S2A) and (ii) the mRNA JAK2V617F mutational burden was 99.5% in JAK2V617F HUVEC (Online Supplementary Figure S2B). Western blot analysis revealed induced pro- tein expression of JAK2 in JAK2V617F- and JAK2WT-trans- duced cells. The introduction of JAK2V617F led to increased phosphorylation of JAK2, STAT3 and AKT, in agreement with hyperactivation of the JAK/STAT pathway (Online Supplementary Figure S2C). Under resting conditions, measurement of thrombin generation at the surface of
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Figure 3. JAK2V617F-expressing endothelial cells have a pro-adhesive phenotype in vitro and in vivo. (A,B) Under static conditions (A) normal mononuclear cells and (B) neutrophils adhere more to human umbilical vein endothelial cells (HUVEC) expressing JAK2V617F than to JAK2WT HUVEC. HUVEC transduced with lentiviruses con- taining green fluorescent protein alone were used as negative controls. Statistical significance determined by one-way analysis of variance and the post-hoc Tukey test. Results are expressed as mean value ± SEM from three experiments. (C,D) Under flow conditions (C) adhesion of neutrophils is increased in the presence of JAK2V617F HUVEC and (D) neutrophil velocity is reduced. Each dot represents one cell. Statistical significance was determined by the Student t-test. Results are expressed as mean value ± SEM from three experiments. (E) Representative images of adhesion of neutrophils on JAK2WT and JAK2V617F HUVEC under flow conditions. (F) Representative images of leukocyte adhesion in mesenteric venules of control and Pdgfb-iCreERT2;JAK2V617F/WT mice treated with tumor necrosis factor (TNF). (G) Leukocyte rolling and (H) adhesion are increased in mesenteric venules of Pdgfb-iCreERT2;JAK2V617F/WT mice treated with TNF. Results are expressed as mean value ± SEM. Statistical significance was determined by the Mann-Whitney test. *P<0.05; ***P<0.001. ****P<0.0001.
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haematologica | 2019; 104(1)