Identification of the Origins and Characterization of the Consequences of Megakaryocytic Hyperplasia in Idiopathic Myelofibrosis.

Megakaryocytic (MK) hyperplasia is a pathological hallmark of the Philadelphia chromosome-negative myeloproliferative disorders. We studied the mechanisms of MK hyperplasia in idiopathic myelofibrosis (IMF) and evaluated the role of MKs in the generation of marrow fibrosis and increased microvessel density in IMF. Isolated CD34+ cells from normal mobilized peripheral blood (NMPB) and peripheral blood from IMF patients (IMFPB) were cultured for 15 days using serum-free media in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO). 5 out of 11 IMF patients were JAK2V617F positive. In the absence of TPO, after 11 days of culture, the percentage of CD41+ cells generated in IMFPB CD34+ cell cultures was 17.3% while only 1.1% of the cells were CD41+ in cultures of NMPB CD34+ cells. The addition of TPO (5ng/ml) led to a 19-fold increase in the percentage of NMPB CD41+ cells as compared to a 1.5 fold increase of IMF CD41+ cells. The absolute numbers of CD41+ cells generated from JAK2V617F positive and negative IMF CD34+ cells in the presence of TPO by day 11 were 5.4 fold and 4 fold greater respectively than normal control CD34+ cells. IMF MKs were smaller and had a lower degree of ploidy as determined flow cytometrically. The percentage of MK undergoing apoptosis was assessed by CD41/propidium iodide (PI) staining and TUNEL assay and revealed that far fewer IMF MKs underwent apoptosis by day 15 (7.7% +/− 1.6(IMF) vs 16.8 +/− 3.2 normal controls, p<0.05). On day 15 CD61+ cells were immunoselected and cultured for an additional 10 days under similar conditions. These cells were evaluated for the expression of the anti-apoptotic protein Bcl-xL, which was present in 40 +/− 8 % of normal MKs by the third day of culture as compared to 79.9 +/− 3 % of IMF MKs (p<0.01) (n=7). Since cytokine production by MKs has been implicated in the generation of marrow fibrosis and increased microvessel density in IMF, we evaluated the production of TGF-beta, VEGF and PDGF in the conditioned media (CM) prepared from NMPB and IMFPB CD34+ and CD61+ cell cultures. On day 11 there was a greater then 10 fold higher concentration of VEGF in IMFPB CD34+ cell CM but the concentration of VEGF was not increased in IMF CD61+ cell CM. The concentration of TGF-beta was 12 fold higher in IMF CD61+ cell CM than normal CD61+ CM. The concentration of PDGF was not increased in IMF CD34+ or CD61+ cells CM. These data indicate that MK hyperplasia can be accounted for by the greater capacity of IMF CD34+ cells to generate MKs, which appears to occur irrespective of the presence of JAK2V617F mutation. Unlike normal megakaryocytopoiesis, the generation of a significant proportion of IMF MKs is independent of TPO. In addition, decreased MK apoptosis, which is a consequence of increased Bcl-xL over-expression in IMF MKs contributes to the development of MK hyperplasia by favoring MK accumulation. IMF MKs likely lead to increased marrow microvessel density and fibrosis by their excessive production of TGF-beta but not PDGF. By contrast the increased VEGF observed in the culture systems appears to be produced by cells other than IMF MKs. The MK hyperplasia in IMF, therefore, represents a pivotal cellular event in IMF, which might serve as a target for future therapeutic interventions.