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.

Reduced expression of TGF beta1RII in agnogenic myeloid metaplasia is not due to mutation or methylation.

Agnogenic myeloid metaplasia (AMM) is characterized by bone marrow fibrosis and enhanced proliferation of megakaryocytes and CD34+ cells. We have analyzed the factors that could lead to reduced expression of TGF beta1RII in CD34+ cells of AMM patients. Our results demonstrate absence of mutations in the coding region and the promoter of this gene and absence of CpG methylation of its promoter in AMM patients. Further studies on transcriptional regulation of TGF beta1RII involving its cis-regulatory elements, the interacting transcription factors and their association with HDAC will provide valuable information on the pathogenesis of AMM and are under current investigation.

TPO/Mpl Studies in Agnogenic Myeloid Metaplasia.

BACKGROUND: Agnogenic myeloid metaplasia (AMM) is one of the Philadelphia chromosome negative myeloproliferative disorder and is diagnosed by hyperplasia of atypical megakaryocytes, hepatosplenomegaly, extramedullary hematopoiesis and bone marrow fibrosis. Fibrosis is considered to be a secondary consequence of enhanced levels of fibrogenic growth factors such as TGF beta1, bFGF and PDGF produced by enhanced numbers of megakaryocytes, while the primary cause is considered to be the enhanced proliferation of a defective stem cell. We have previously reported that thrombopoietin (TPO) is elevated in patients with AMM. Others have reported that Mpl protein is decreased in these patients. Since TPO is essential for the development of megakaryocytes, and Mpl protein is the receptor for TPO, we extended the study of TPO/Mpl to in vitro and in vivo cell culture systems to better understand the mechanism that leads to reduced Mpl protein in AMM patients. RESULTS: Plasma TPO levels were significantly elevated and Mpl protein levels were significantly reduced in AMM patients in concordance with previous studies. Platelet Mpl transcripts in AMM were however similar to those in controls. We also cloned Mpl cDNA from AMM patients and tested for their ability to make functional proteins in vitro and in the in vivo system of 293 T human embryonic kidney cells. Their expression including the glycosylated forms was similar to those from the controls. We also measured the level of translation initiation factor, eIF4E and found it to be increased in patients with AMM demonstrating that the reduced Mpl protein may not be due to translation defects. CONCLUSIONS: Our studies using the in vitro and in vivo systems further confirm that reduced Mpl protein levels are not due to defects in its transcription/translation. Reduced Mpl protein could be due to its increased internalisation owing to enhanced plasma TPO or in vivo intrinsic defects in patients with AMM.

Osteosclerosis in idiopathic myelofibrosis is related to the overproduction of osteoprotegerin (OPG).

OBJECTIVE: The aim of this study is to investigate the mechanism of osteosclerosis in IMF in relation to OPG derangement. METHODS: Plasma OPG level was assayed by OPG ELISA in 19 patients with IMF, 15 patients with other myeloproliferative disorders (MPDs), and 12 normal volunteers as controls and correlated with the degree of osteosclerosis. Furthermore, the level of OPG mRNA, in the cultured bone marrow stromal (BMS) cells of patients with IMF and anemia patients used as controls, in the presence or absence of TGF-beta1, was studied by real-time RT-PCR. RESULTS: The present study showed that blood OPG level was significantly elevated in patients with IMF as compared to patients with other MPDs (p < 0.01) or normal volunteer controls (p < 0.05), and there was no significant difference in the level between patients with MPDs and controls. In addition, there was a positive correlation (r=0.67, p=0.04) between plasma OPG levels and the degree of osteosclerosis. There was no difference in the OPG mRNA in patients with IMF as compared with controls even on TGF-beta1 stimulation. CONCLUSION: These results suggest that osteosclerosis in IMF may be related to overproduction of OPG and enhanced level of OPG is not due to the effect of TGF-beta1 on the BMS cells. It could be due to the effect of TGF-beta1 or other growth factors on cells other than BMS cells such as the osteoblasts.

Plasma matrix metalloproteinase and tissue inhibitor of metalloproteinase in patients with agnogenic myeloid metaplasia or idiopathic primary myelofibrosis.

Agnogenic myeloid metaplasia (AMM) is characterized by bone marrow fibrosis with abnormal accumulation of extracellular matrix components (ECM), which is dependent on the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Twenty-five patients with AMM, 30 with essential thrombocythemia (ET), 12 with polycythemia vera (PV) and 20 normal control subjects were studied. AMM patients had decreased plasma levels of MMP-3 and marked elevated levels of TIMP-1, but MMP-1, MMP-2 and MMP-9 levels were not significantly different from control subjects. Elevated levels of plasma TIMP-1, but not MMPs, were found in ET and PV. Reduced MMP activity together with increased TIMP-1 activity may be essential in fibrosis formation.

Hypermethylation of the P15INK4b and P16INK4a in agnogenic myeloid metaplasia (AMM) and AMM in leukaemic transformation.

Hypermethylation of p15 and p16 genes was determined in 32 patients with agnogenic myeloid metaplasia(AMM), also known as idiopathic myelofibrosis (MF). These included 10 patients in leukaemic transformation phase. Using polymerase chain reaction-based methylation analysis assay methods, with substantiation using Southern blot analysis, the study showed no hypermethylation of p15 or p16 genes in the chronic phase of AMM, but p15 gene hypermethylation was found in four patients (40%) and p16 gene hypermethylation in two patients (20%) when they were in leukaemic transformation stage. Furthermore, two of the patients in leukaemic transformation were found to have both p15 and p16 gene hypermethylation, demonstrating possible multiple gene hypermethylation in the same patient. Thus, hypomethylation agents for treating patients with AMM in leukaemic transformation may be appropriate for future trials.