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JAK2V617F endothelial cells are pro-thrombotic
Arterial and venous thromboses are the main causes of morbidity in Philadelphia chromosome-negative MPN with reported incidences ranging from 12-39% in poly- cythemia vera and 11-25% in essential thrombocythemia.7 The pathogenesis of thrombosis in patients with MPN is complex and still largely elusive.8 A variety of blood cells have been reported to participate in the pathophysiology of thrombosis in these neoplasms: (i) platelets isolated from MPN patients show signs of enhanced in vivo activation;9 (ii) leukocytes are activated and hyperleukocytosis is an independent risk factor for thrombosis;9-11 and (iii) red blood cells from patients with polycythemia vera display increased adhesion to the endothelium.12 However, there is evidence that JAK2V617F can be present not only in blood cells but also in endothe- lial cells (EC) from JAK2V617F-positive MPN patients.13-15
Under physiological conditions, the endothelium main- tains a hemostatic balance between pro-thrombotic and anti-thrombotic factors. When stimulated by extrinsic fac- tors such as inflammatory cytokines, hypoxia or antiphos- pholipid antibodies, EC become activated and promote thrombosis.16 Whether EC can become pro-thrombotic due to intrinsic modifications such as genetic mutations, has not yet been demonstrated.
In the current study, we tested whether vascular EC expression of JAK2V617F is sufficient to promote a pro- thrombotic state. Specifically, we investigated the hemo- static properties of JAK2V617F-expressing EC (hereafter, simply JAK2V617F EC) and these cells’ role in thrombus for- mation using human EC overexpressing human JAK2V617F, and mice with EC-specific JAK2V617F expression.
Methods
In vitro static adhesion of normal blood cells on endothelial cells
Blood from healthy volunteers was collected into test-tubes containing EDTA after informed consent had been obtained. Mononuclear cells and neutrophils were isolated by Pancoll densi- ty gradient or neutrophil separation medium (Polymorphprep, Fresenius Kabi) and marked with CellTracker Orange. Cells (105) were plated over confluent human umbilical vein endothelial cells (HUVEC) for 1 h at 37°C. After removal of non-adherent cells, the adherent cells were visualized using a fluorescence microscope (AxioObserver, Zeiss), and images were analyzed by ZEN imaging software (Zeiss). Experiments were performed using three to six wells per condition with 12 images taken in each well. The method of quantification has been described elsewhere15,17 Where noted, we added 10 mg/mL P-selectin blocking antibody (AK4 clone, BioLegend) 30 min before plating blood cells on HUVEC.
In vitro neutrophil adhesion on human umbilical vein endothelial cells under flow conditions
Channels (Vena8 Endothelial + Cellix) were coated with human fibronectin (100 ng/mL) before infusing 3x106/mL HUVEC.18 After 2 h at 37°C, cells were exposed for 48 h to two-period flow (Kima Pump, Cellix) with alternating perfusion for 3 min (at 600 mL/min) and resting for 20 min (no flow). After 48 h, a conflu- ent monolayer had developed. Neutrophils (3x106/mL) were infused with a 10 mL/min venous flux and imaged using a Zeiss microscope, 20X in phase contrast, for 5 min. Channels were washed with medium and images of every channel were record- ed. Neutrophil quantification was performed blindly and the neu- trophil velocity was studied with FIJI (Image J).
In vivo leukocyte adhesion to mesenteric venules Pdgfb-iCreERT2;JAK2V617F/WT mice and Pdgfb-iCreERT2;JAK2V617F/WT mice were used 15-20 days after tamoxifen injection. Mice were injected, intraperitoneally, with 250 ng tumor necrosis factor-alpha (TNF-α) and, 4h later, anesthetized with ketamine/xylazine. Leukocytes were stained with 3.6 mg/kg Rhodamine 6G (Sigma- Aldrich). For each mouse, five mesenteric venules of 150 to 250 μm diameter were observed for 90 sec within 30 min after the sur- gical procedure, using a fluorescent microscope (AXIO Zoom.V16, Zeiss). Where noted, 25 mg of P-selectin blocking antibody (RB40 clone, BD Biosciences) or isotype control (A110-1 clone, BD
Biosciences) were injected 5 min before starting the analysis.
Mouse model of thrombosis
To induce platelet activation, the mice were given an intraperi- toneal injection of 75 μg/kg collagen (Nycomed Pharma) and 30 mg/kg epinephrine (Helena Laboratories) 3 min before euthana- sia.19,20 To increase inflammation, 250 ng TNF-α (RD Systems) were injected 4 h before the animals were sacrificed. After euthanasia, intracardiac puncture was performed and phosphate- buffered saline was perfused for 3 min, followed by 10% formalin for another 3 min. Where noted, 25 mg P-selectin blocking anti- body were injected 4 h prior to euthanasia.
Statistics
Results are expressed as the mean ± standard error of mean (SEM). Statistical significance was calculated using the Student t test or Mann-Whitney statistical test to compare differences between two groups. For multiple groups, we used one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test or two-way ANOVA followed by the Sidak post-hoc test. GraphPad Prism 6 software was used. A P value <0.05 was con- sidered statistically significant.
Animals
Animal experiments were performed in accordance with the guidelines provided by the Institutional Animal Care and Use Committee at Inserm (Agreement number C45-234-6).
Results
Pdgfb-iCreERT2;JAK2V617F/WT mice are a reliable model to investigate JAK2V617F endothelial cell properties
To analyze the specific role of JAK2V617F EC in thrombus formation, we crossed Pdgfb-iCreERT2 mice with condi- tional flexed JAK2 (JAK2V617F) mice,21 to allow heterozy- gous expression of JAK2V617F in EC after tamoxifen admin- istration. Since oral tamoxifen administration has been shown to result in recombination activity in megakary- ocytes within 2 days,22 all experiments were performed and analyzed 15-20 days after tamoxifen injection, to allow time for recombined megakaryocytes to mature and be cleared. We first analyzed the efficiency of recombina- tion using Pdgfb-iCreERT2;mT/mG mice, and observed that all vessels in the mesentery and retina were positive for green fluorescent protein (GFP) (Online Supplementary Figure S1A-C). To confirm EC JAK2V617F expression in Pdgfb-iCreERT2;JAK2V617F/WT mice, we sorted CD31-positive cells from the kidney and observed a 50% ratio of JAK2V617F:total JAK2 in accordance with a heterozygous expression of JAK2V617F in all EC (data not shown). In sorted CD31-positive lung EC, western blot analysis demonstrat- ed an increased level of phosphorylation of the down- stream effector AKT in line with increased activation of
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