Quantitative analysis of growth factor production in the mechanism of fibrosis in agnogenic myeloid metaplasia.

OBJECTIVE: The current study quantified the growth factors in megakaryocytes and monocytes and correlated them to the degree of fibrosis, as there is no quantitative analysis of growth factors from megakaryocytes or monocytes reported in patients with agnogenic myeloid metaplasia (AMM). MATERIALS AND METHODS: Megakaryocytes were obtained from cultured blood CD34+ cells. CD14+ cells were sorted by magnetic cell sorting. Quantitative analyses of the growth factors were obtained by real-time reverse-transcriptase polymerase chain reaction techniques and enzyme-linked immunobsorbent assay (ELISA). RESULTS: 1) We found that mRNA levels of transforming growth factor (TGF) beta1, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) produced by the megakaryocytes were significantly elevated in AMM compared with those in normal controls (p < 0.05). Although these growth factors were elevated severalfold in AMM compared with other myeloproliferative disorders (MPDs) including essential thrombocythemia and polycythemia vera, they were not statistically significant. 2) TGF-beta1 was more abundantly produced than PDGF or FGF. 3) The mRNA levels of these growth factors produced from CD14+ cells were not significantly elevated in AMM compared with other MPDs or controls; the AMM mRNA levels were significantly elevated only in some patients. 4) The correlation of mRNA levels of these growth factors with the degree of myelofibrosis in AMM was significant with megakaryocytes (r = 0.73) but not with monocytes (r = 0.23). 5) ELISA of the growth factors from the cultured megakaryocytes showed that in most of the patients with AMM and other MPDs, and in volunteer controls, the growth factors were undetectable, and only a few patients with AMM (three each of TGF-beta1 and PDGF and one of FGF) and other MPDs (two of TGF-beta1 and one each of PDGF and FGF) had significantly elevated protein levels of these growth factors. CONCLUSIONS: 1) In AMM, the mRNA levels of these fibrosing growth factors are significantly elevated in megakaryocytes, and they were only elevated in a few patients in the monocyte-macrophage lineages. 2) mRNA of TGF-beta1 is more abundantly produced than that of PDGF or FGF from megakaryocytes. 3) A statistically significant correlation between the growth factor mRNA levels with the degree of myelofibrosis in AMM suggests that these fibrosing growth factors produced by the megakaryocytes may be associated with the etiology of bone marrow fibrosis in AMM. 4) Failure to substantiate at the protein level that megakaryocytes are the main source of growth factors production suggests that other factors or cells initiating translation of the growth factors in the megakaryocytes may also be important in the process of bone marrow fibrosis in AMM. Further studies are necessary.

Duke Scholars

Author Amit Goldberg Medicine, Hematology