Clonal Origin of Circulating Endothelial Progenitor Cells in Patients with Myelofibrosis with Myeloid Metaplasia.

Chronic myeloproliferative diseases are clonal disorders of the hematopoietic stem cell. Among these conditions myelofibrosis with myeloid metaplasia (MMM) is characterized by increased numbers of circulating CD34+ cells compared to other Ph-negative myeloproliferative disorders and normal controls. Increased numbers of circulating CD34+ cells are likely due to mobilization of primitive hematopoietic stem cells/progenitor cells (HSC/HPC) from the bone marrow of patients with MMM following activation of proteases and disruption of adhesion interactions, which normally result in the retention of HSC/HPC within the marrow. A more detailed characterization of circulating CD34+ cells in MMM suggested that a cell fraction which included VEGFR-2+, CD34+VEGFR-2+, CD133+, and CD34+CD133+ cells contained endothelial progenitor cells.

In the present work we cultured peripheral blood mononuclear cells (PBMNC) from three patients with MMM (one male and two females), from one female patient with essential thrombocythemia (ET) and from three normal females in the presence of fibronectin and vascular endothelial growth factor (VEGF) for 7 days, in order to obtain endothelial cell colonies. The endothelial origin of colonies was assessed by VE-cadherin and CD31 staining. Clonality of endothelial cell colonies was evaluated in female subjects by X-chromosome inactivation studies based on determination of differential DNA methylation at the two alleles of X-linked polymorphic loci (Humara or PGK). Such evaluation was performed on at least 8 single colonies and on a pool of 7–8 colonies from each subject. The male patient with MMM had a 7q34 deletion in 50 % of metaphases. The deletion resulted in loss of heterozygosity at the polymorphic marker D7S684 which was therefore evaluated on both endothelial and hematopoietic colonies grown from PBMNC. Normal subjects showed a polyclonal pattern of X-chromosome inactivation in endothelial colonies, whereas MMM and the ET female patients showed selective inactivation of the same chromosome X which was inactivated in clonal peripheral blood polymorphonuclear cells. Loss of heterozygosity at the polymorphic marker D7S684 was observed in 6/10 and 4/9 endothelial and hematopoietic colonies, respectively, from the male patient with the 7q34 deletion. Our results suggest that in MMM, and possibly in other chronic myeloproliferative diseases, circulating endothelial progenitor cells are clonal and the disease is due to transformation of a hematopoietic stem cell which also retains endothelial differentiation potential.