Page 88 - 2019_01-Haematologica-web

A. Guy et al.
trophil adhesion on JAK2V617F HUVEC pretreated with hydroxyurea (Figure 7D). Having previously demonstrat- ed that the pro-adhesive phenotype of JAK2V617F EC was mediated by P-selectin, we examined whether the protec- tive effect of hydroxyurea acted through reduction of endothelial P-selectin. As we had previously shown that soluble P-selectin was a reflection of endothelial P-selectin in Pdgfb-iCreERT2;JAK2V617F/WT mice, we measured levels of soluble P-selectin after treatment with hydroxyurea. We observed that the P-selectin:platelet ratio was significantly lower in hydroxyurea-treated Pdgfb-iCreERT2;JAK2V617F/WT mice than in untreated mice (Figure 7E). In these same mice, we used immunostaining to quantify P-selectin expression at the surface of carotid EC. We observed a sig-
nificant decrease of endothelial membrane P-selectin expression, confirming a direct effect of hydroxyurea on JAK2V617F EC (Figure 7F). This effect is in part secondary to JAK/STAT pathway inhibition, as hydroxyurea adminis- tration reduces the level of STAT3 phosphorylation in JAK2V617F HUVEC (Online Supplementary Figure S6). Finally, we observed decreased vWF concentrations in the super- natant of JAK2V617F HUVEC treated with hydroxyurea, indicating a reduced release of Weibel Palade bodies (Figure 7G). Collectively, in vitro and in vivo results demonstrate that hydroxyurea has a direct effect on JAK2V617F EC, decreasing endothelial P-selectin release and surface expression, thus decreasing the pro-thrombotic phenotype of the EC.
ABC
D
EFG
Figure 7. Treatment with hydroxyurea decreases the pro-thrombotic and pro-adhesive phenotype of JAK2V617F-expressing endothelial cells in vitro and in vivo via inhibiting P-selectin and von Willebrand factor expression. (A) Treatment with hydroxyurea for 15 days decreases tumor necrosis factor-induced thrombosis in the lungs of Pdgfb-iCreERT2;JAK2V617F/WT mice. Treatment with hydroxyurea decreases (B) rolling and (C) adhesion of leukocytes on mesenteric venules in Pdgfb-iCreERT2;JAK2V617F/WT mice treated with tumor necrosis factor. Results are expressed as mean value ± SEM. Statistical significance was determined by the Mann-Whitney test. *P<0.05, ***P<0.001. (D) Pre-treatment of JAK2V617F HUVEC with hydroxurea (HU) decreases static adhesion of neutrophils. Results are expressed as mean value ± SEM. Statistical significance determined by two-way ANOVA analysis of variance and the Sidak post-hoc test. (E) Treatment with hydrox- yurea for 15 days led to a decrease of the ratio of soluble P-selectin:number of platelets in plasma of Pdgfb-iCreERT2;JAK2V617F/WT mice treated with tumor necrosis factor. (F) Hydroxyurea decreases the expression of P-selectin at the surface of carotid JAK2V617F endothelial cells. Each dot represents one image with four images per mouse (n=4). (G) Treatment of JAK2V617F HUVEC with hydroxyurea decreases secretion of von Willebrand factor (vWF). Results are expressed as mean value ± SEM. Statistical significance was determined by the Mann-Whitney test. *P<0.05.
78
haematologica | 2019; 104(1)