JAK2V617F - Positive Endothelial Cells Display Pro-Thrombotic Characteristics
Authors: Guadall Anna , Lesteven Elodie , Letort Gil , Awan Toor Sarah , Pognant Doriane , Brusson Megane , Verger Emmanuelle , Larghero Jerome , Vanneaux Valerie , Chomienne Christine , El Nemer Wassim , Cassinat Bruno , Kiladjian Jean-Jacques 
Published: October 2019

Background: Clinical complications of BCR-ABL1-negative Myeloproliferative Neoplasms (MPNs) include mainly vascular events and on longer term transformation to Myelofibrosis (MF) or acute leukemia. MPNs are also associated with neo-angiogenesis leading to extramedullary hematopoiesis and increased vascularity in the spleen and bone marrow. The most frequent molecular alteration present in hematopoietic cells is the JAK2V617F mutation, which has also been detected in hepatic Endothelial Cells (ECs) of MPN patients with Budd-Chiari syndrome (BCS) and in ECs derived from patients' splenic capillaries. The consequences of the presence of the JAK2V617F mutation into the endothelial lineage are not known although these cells play an important role in thrombosis.

Methods: Isogenic JAK2 wild-type (WT) and JAK2V617F mutant induced pluripotent stem cells (iPSC) were derived from an MPN patient. The reprogrammed clonal cells were induced to differentiate toward mesoderm for 3 days and then driven toward the endothelial lineage using Stem-Pro medium, Vascular Endothelial Growth Factor (VEGF) and Forskolin. Enriched CD34+/CD144+ progenitors were then matured in VEGF-supplemented Stem-Pro medium for 5 days before being cultured in the EGM-2 specific EC medium in the presence of the TGF-β inhibitor SB-431542. Cells were harvested and tested for specific surface markers by flow cytometry before expanding them; passages 3 to 6 were used for further characterization and functional studies. Transcriptomic analysis was performed in triplicate from two independent endothelial differentiation experiments using Affymetrix HTA 2.0 gene expression arrays.

Results: Phenotypically, the endothelial-like cells obtained after differentiation from both mutant and WT JAK2 iPSC displayed a classical EC morphology and were positive for CD34 and EC markers (CD144, CD309, CD31 and ICAM-1). Such characteristics were conserved for at least 6 rounds of cell passaging. TNF-α treatment increased ICAM-1 cell surface levels and induced the expression of VCAM-1, as observed in other EC models. Our iPS-derived cells were also responsive to VEGF, which induced cellular proliferation and angiogenesis demonstrated by tube formation in Matrigel with both JAK2V617F and WT ECs. As expected, JAK2V617F ECs exhibited higher levels of phosphorylated forms of JAK2, STAT3 and Akt in a serum and growth factor-reduced medium, showing that, as in hematopoietic cells, the presence of the mutation increases basal intracellular signaling. Interestingly, Weibel-Palade bodies detection by confocal microscopy revealed a more important amount of Von Willebrand factor (vWF) in JAK2V617F ECs (2-fold increase), suggesting a higher pro-thrombotic potential for these cells. Basal surface expression of the adhesion molecule ICAM-1 was also clearly increased (1.8-fold) compared to WT ECs suggesting that JAK2V617F ECs may be more prone to recruit activated leukocytes expressing LFA-1, the ICAM-1 receptor. Gene expression microarrays analysis confirmed a significantly higher expression of cell adhesion molecules involved in leukocyte or platelet adhesion in JAK2V617F vs. WT ECs (ICAM-1, VCAM-1, or P-selectin), together with several inflammatory chemokines (IL8, CXCL1, or CXCL5), chemokine receptors (CXCR4 or IL7R) and extracellular matrix modulators (Periostin, Tenascin C, Lysyl Oxidase, or MMP-10) . Finally, Gene Set Enrichment Analysis pointed out gene sets induced in situations of inflammation, response to cytokines and hypoxia. These results could be in agreement with an activated endothelium, leukocyte recruitment and vascular remodeling.

Conclusion: Previous discovery of JAK2V617F-positive endothelial cells in the splanchnic vasculature of patients with BCS or MF was of particular interest because of the well-known association between thrombosis and MPN. In this work we first demonstrate that the presence of JAK2V617F doesn't impair endothelial differentiation from stem/progenitor cells. Moreover, JAK2V617F-mutant ECs display features of pro-inflammatory and pro-thrombotic EC activation, which we are currently further characterizing with additional functional studies. Finally, this iPSC model may represent an effective screening tool for molecules antagonizing the pro-thrombotic events in a JAK2V617F context.

Disclosures

Kiladjian: Novartis: Honoraria, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.