Thrombosis is the main cause of morbidity and mortality in patients with JAK2V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with MPN. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrate that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, we provide evidence that JAK2V617F endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can impact the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause which account for 50% of recurrent venous thrombosis.

Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y ₂ ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. The blockade of osteopontin or its integrin receptors α _v β ₃ /β ₅ by specific antibodies or antagonists inhibited UTP-induced migration. Moreover, the blockade of ERK-1/ERK-2 MAP kinase or rho protein pathways led to the inhibition of both UTP-induced osteopontin increase and migration, demonstrating the central role of osteopontin in this process. Taken together, these results suggest that extracellular nucleotides, and particularly UTP, can induce arterial SMC migration via the action of osteopontin.

Background—: CXC-chemokine receptor 4 (CXCR4) regulates the retention of stem/progenitor cells in the bone marrow (BM), and the CXCR4 antagonist AMD3100 improves recovery from coronary ligation injury by mobilizing stem/progenitor cells from the BM to the peripheral blood. Thus, we investigated whether AMD3100 also improves recovery from ischemia/reperfusion injury, which more closely mimics myocardial infarction in patients, because blood flow is only temporarily obstructed. Methods and Results—: Mice were treated with single subcutaneous injections of AMD3100 (5 mg/kg) or saline after ischemia/reperfusion injury. Three days later, histological measurements of the ratio of infarct area to area at risk were smaller in AMD3100-treated mice than in mice administered saline, and echocardiographic measurements of left ventricular function were greater in the AMD3100-treated mice at week 4. CXCR4 ⁺ cells were mobilized for just 1 day in both groups, but the mobilization of sca1 ⁺ /flk1 ⁺ cells endured for 7 days in AMD3100-treated mice compared with just 1 day in the saline-treated mice. AMD3100 upregulated BM levels of endothelial nitric oxide synthase (eNOS) and 2 targets of eNOS signaling, matrix metalloproteinase-9 and soluble Kit ligand. Furthermore, the loss of BM eNOS expression abolished the benefit of AMD3100 on sca1 ⁺ /flk1 ⁺ cell mobilization without altering the mobilization of CXCR4 ⁺ cells, and the cardioprotective effects of AMD3100 were retained in eNOS-knockout mice that had been transplanted with BM from wild-type mice but not in wild-type mice with eNOS-knockout BM. Conclusions—: AMD3100 prolongs BM progenitor mobilization and improves recovery from ischemia/reperfusion injury, and these benefits appear to occur through a previously unidentified link between AMD3100 and BM eNOS expression.