Patients with myeloproliferative disorders (MPD) are at a high risk of developing thrombotic events. We hypothesize that one of the contributory factors to this thrombotic tendency is the involvement of vascular endothelial cells (EC) by the malignant process. In vitro and in vivo assays were used to determine the involvement of EC in patients with MPD. Endothelial progenitor cells (EPC) were assayed from the peripheral blood (PB) mononuclear cells (MNCs) of 3 normal controls (NC) and 16 patients with MPD (12 polycythemia vera (PV), 4 primary myelofibrosis (PMF). MNC were cultured for 2 days in EC growth media on fibronectin(FN)-coated plates. The non-adherent cells were then harvested and transferred to a secondary FN-coated plate for additional 5–14 days. EC colonies were identified by their morphological appearance. The colonies were plucked and analyzed for PECAM-1(CD31), VE-Cadherin(CD144), VEGFR-2, vWF, Endoglin(CD105), ULEX-1, CD45, CD14 by flow cytometry and acetylated LDL(Ac-ADL) uptake. EC colonies were CD31+CD144+VEGFR2+ULEX-1+vWF+CD105+CD45+CD14+ and capable of taking up Ac-LDL and when exposed to TNF-α and IL-1β, expressing ICAM(CD54) and E-selectin(CD62e). MPD MNC formed fewer numbers of EC colonies than normal MNC (31.1±34.2 vs 78.8±28.9; p<0.01) and required more prolonged periods of culture (14 vs 5days). MPD EC colonies were also analyzed for JAK2V617F(JAK2VF) by nested-PCR. 74.6% of MPD EC colonies were homozygous(homo) JAK2VF, 14.9% were heterozygous(hetero) JAK2VF and 10.4% were wild type(wt) JAK2. Interestingly, MNCs from JAK2VF−MPD(148±47) formed greater numbers of EC colonies than NC MNC (78.8±28.9; p≤0.01). MNC from patients with a high burden of JAK2VF alleles (10.3±18.5; p<0.01) formed fewer EC colonies than NC or patients with a low burden of JAK2VF (65.9±28.15; p≤0.01). These EPC assayed in vitro which produced cells with both myeloid and endothelial markers are likely due either to contamination with JAK2VF myeloid cells or the result of the transdifferentiation of myeloid progenitor cells into EC (
Disclosure: No relevant conflicts of interest to declare.