transforming-growth-factor-beta and Thrombocythemia--Essential

The BCR-ABL-negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), entered the spotlight in 2005 when the unique somatic acquired JAK2 V617F mutation was described in >95% of PV and in 50% of ET and PMF patients. For the very rare PV patients who do not harbor the JAK2 V617F mutation, exon 12 JAK2 mutants were discovered also to result in activated forms of JAK2. A minority of ET and PMF patients harbor mutations that constitutively activate the thrombopoietin receptor (TpoR). In bone marrow reconstitution models based on retroviral transduction, the phenotype induced by JAK2 V617F is less severe and different from the rapid fatal myelofibrosis induced by TpoR W515L. The reasons for these differences are unknown. Exactly by which mechanism(s) one acquired somatic mutation, JAK2 V617F, can promote three different diseases remains a mystery, although gene dosage and host genetic variation might have important functions. We review the recent progress made in deciphering signaling anomalies in PV, ET and PMF, with an emphasis on the relationship between JAK2 V617F and cytokine receptor signaling and on cross-talk with several other signaling pathways.

Topics: Animals; Disease Models, Animal; Hematopoietic Stem Cells; Humans; Janus Kinase 2; Mutation; Phosphorylation; Polycythemia Vera; Primary Myelofibrosis; Receptors, Erythropoietin; Receptors, Granulocyte Colony-Stimulating Factor; Receptors, Thrombopoietin; Signal Transduction; STAT Transcription Factors; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Thrombocythemia, Essential; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Although bone marrow fibrosis is a lethal condition, its underlying mechanism is not fully understood. This study aimed to investigate the pathogenesis of fibrosis in the bone marrow through histologic examination of mast cell infiltration and the expression of fibrosis-associated cytokines. We analyzed 22 bone marrows with fibrosis (8 primary myelofibrosis [PMF], 5 post-essential thrombocythemia [ET], myelofibrosis, and 9 myelodysplastic syndrome [MDS] with bone marrow fibrosis [BMF]). Immunohistochemical and immunofluorescence stainings were performed using anti-mast cell tryptase, interleukin (IL) 13, transforming growth factor β (TGF-β), CD34, and CD42b antibodies. The number of mast cells in bone marrows with fibrosis was significantly higher than that in controls (P<.0001 for all cases with fibrosis versus control, P=.0470 for PMF versus control, P<.0001 post-ET myelofibrosis versus control, and P=.0005 for MDS with BMF versus control). Moreover, bone marrows with higher fibrotic grades exhibited greater amounts of infiltrating mast cells. Mast cells were positive for TGF-β and IL-13 in bone marrows with fibrosis of all 3 groups. Megakaryocytes were negative for TGF-β in post-ET and MDS with BMF, but some megakaryocytes in PMF were weakly positive for TGF-β. Megakaryocytes were negative for IL-13 in all 3 groups. Blasts were negative for both TGF-β and IL-13 in all 3 groups. Thus, TGF-β- and IL-13-producing mast cells might be key players in the development of BMF. Therefore, mast cells could be potential therapeutic targets for the treatment of BMF.

Topics: Aged; Aged, 80 and over; Bone Marrow; Case-Control Studies; Female; Fluorescent Antibody Technique; Humans; Interleukin-13; Male; Mast Cells; Megakaryocytes; Middle Aged; Myelodysplastic Syndromes; Primary Myelofibrosis; Thrombocythemia, Essential; Transforming Growth Factor beta

The development of bone marrow fibrosis and thrombosis are main causes of morbidity in essential thrombocythemia (ET). Monocyte activation has been associated to the production of fibrosis-related cytokines and pro-thrombotic factors. The aim of this study was to identify new markers of monocyte activation in Phi-negative myeloproliferative neoplasms and to search for their relationship with clinical features. Forty-five patients comprising 30 ET, eight myelofibrosis and seven polycythemia vera were included. We evaluated the alpha subunit of IL-2 receptor (CD25) on monocytes, basal and LPS-induced IL-1beta release from mononuclear cells, and monocyte TGF-beta mRNA content. Patients who had thrombotic events displayed higher monocyte CD25 levels (6.2%) than those without symptoms (1.3%) and controls (2.6%), p=0.0006. JAK2V617F-positive patients had higher monocyte CD25 expression levels (4.7%), than JAK2V617F-negative (2.6%), p=0.0213. Patients with myeloproliferative neoplasms had similar monocyte CD25 expression than controls, both, in basal conditions and after cell adhesion. IL-1beta release and TGF-beta mRNA levels were normal. In conclusion, increased monocyte CD25 expression is associated with history of thrombosis and is also up-regulated in patients harboring JAK2V617F mutation. The finding of increased CD25 levels together with normal IL-1beta and TGF-beta production reveals a selective monocyte activation profile in myeloproliferative neoplasms.

Topics: Adult; Aged; Amino Acid Substitution; Case-Control Studies; Female; Humans; Interleukin-1beta; Interleukin-2 Receptor alpha Subunit; Janus Kinase 2; Male; Middle Aged; Monocytes; Mutation; Myeloproliferative Disorders; Thrombocythemia, Essential; Thrombosis; Transforming Growth Factor beta; Young Adult

We discuss a patient with Henoch-Schonlein Purpura (HSP) and a thrombocythaemia which was diagnosed as a coincidental Essential Thrombocythaemia. We suggest that deficiencies in Smad4 expression may allow for escape thrombocythaemia under the influence of the high levels of TGF-beta found in HSP. With normal Smad4 expression TGF-beta provides inhibition of thrombocyte proliferation. While this needs further elucidation, it could lead to a new approach to classification and management of HSP.

Topics: Biopsy; Blood Platelets; Cell Proliferation; Dermatologic Surgical Procedures; Female; Follow-Up Studies; Humans; IgA Vasculitis; Immunosuppressive Agents; Middle Aged; Mycophenolic Acid; Platelet Count; Prednisolone; Skin; Smad4 Protein; Thrombocythemia, Essential; Time Factors; Transforming Growth Factor beta; Treatment Outcome

Plasmatic levels of PDGF-AB, TGFbeta1, and bFGF are increased in patients with essential thrombocythemia (ET) while intraplatelet levels are low for PDGF, normal for TGFbeta, and elevated for bFGF. To evaluate the contribution of gene expression to the dysregulated cytokine levels, we studied platelet PDGF-A, PDGF-B, TGFbeta1, and bFGF mRNA in ET patients before and during anagrelide treatment. We found decreased PDGF-A and PDGF-B, increased TGFbeta1, and normal bFGF mRNA levels. During treatment, mRNA levels remained decreased for PDGF-A, were increased for PDGF-B and normal for TGFbeta1. In untreated patients, protein expression of PDGF paralleled its mRNA levels while different patterns of RNA and protein were found for TGFbeta1 and bFGF.

Topics: Blood Platelets; Fibroblast Growth Factor 2; Growth Substances; Humans; Middle Aged; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Quinazolines; RNA, Messenger; Thrombocythemia, Essential; Transforming Growth Factor beta

Transforming growth factor beta-1 (TGF beta-1) is a potent inducer of fibrosis and has been shown to be essential for the development of bone marrow fibrosis in an animal model of idiopathic myelofibrosis (IMF). IMF belongs to the Philadelphia chromosome negative chronic myeloproliferative disorders (Ph(-) CMPD). Megakaryocytes and platelets have been suggested as the major cellular source of TGF beta-1 in IMF. The osteoclastogenesis inhibitory factor osteoprotegerin (OPG) seems to be regulated by TGF beta-1 and substantial involvement of OPG expression in the process of osteosclerosis in IMF has recently been suggested. In order to determine TGF beta-1 expression in IMF and other Ph(-) CMPD, total bone marrow cells as well as laser-microdissected megakaryocytes were quantitatively analysed by real-time RT-PCR. OPG mRNA expression in fibrotic IMF was correlated with TGF beta-1 mRNA expression in a case-specific manner. Both OPG and TGF beta-1 were detected immunohistochemically in order to delineate cellular origin. When total bone marrow cells were investigated, TGF beta-1 mRNA expression was increased in some but not all cases of IMF (n = 21), with highest values in fibrotic cases. Unexpectedly, increased values were also observed in essential thrombocythaemia (ET, n = 11) when compared to non-neoplastic haematopoiesis (n = 38). Megakaryocytes isolated by laser microdissection displayed elevated TGF beta-1 mRNA levels in most of the CMPD samples with no significant differences discernible between fibrotic IMF, polycythaemia vera (PV) and ET. TGF beta-1 protein was predominantly expressed by the myeloid lineage in Ph(-) CMPD and non-neoplastic haematopoiesis, which, however, displayed lower expression. IMF cases with advanced fibrosis concomitantly overexpressed TGF beta-1 and OPG. Immunohistochemically, OPG expression was found in different stromal cells and a subfraction of megakaryocytes. In conclusion, enhanced TGF beta-1 expression occurs in megakaryocytes as well as myeloid cells in Ph(-) CMPD. TGF beta-1 may be necessary, but is not sufficient, to induce bone marrow fibrosis in IMF because non-fibrotic Ph(-) CMPD entities share this feature with IMF and cannot be discriminated from each other on the basis of TGF beta-1 expression.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Marrow Cells; Chronic Disease; Diagnosis, Differential; Female; Gene Expression; Glycoproteins; Hematopoiesis; Humans; Leukemia, Myeloid, Chronic, Atypical, BCR-ABL Negative; Male; Megakaryocytes; Microdissection; Middle Aged; Myeloproliferative Disorders; Osteoprotegerin; Polycythemia Vera; Primary Myelofibrosis; Protein Array Analysis; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thrombocythemia, Essential; Transforming Growth Factor beta; Transforming Growth Factor beta1

Essential thrombocythaemia (ET) is characterized by the abnormal and sustained proliferation of megakaryocytes. The mechanism for this lineage-specific expansion in ET, remains unclear. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is involved in negative feedback regulation of megakaryopoiesis in both healthy volunteers (HV) and patients with idiopathic thrombocytopenic purpura (ITP). The present study found that megakaryocyte colony-forming units (CFU-MK) of ET patients were less sensitive to TGF-beta1 than those of HV. The expression of Smad4 (Sma- and Mad-related protein-4) in CFU-MK of ET patients was reduced in comparison with that of HV. Finally, to confirm that the impaired TGF-beta1 sensitivity was caused by reduced expression of Smad4, we examined Smad4-transfected CFU-MK from ET patients in the presence of TGF-beta1, and verified that the transfectants were indeed as susceptible as CFU-MK from HV to TGF-beta1. Thus it was surmised that one of the mechanisms for impaired sensitivity of CFU-MK to TGF-beta1 is the reduced expression of Smad4.

Topics: Adult; Aged; Aged, 80 and over; DNA-Binding Proteins; Dose-Response Relationship, Drug; Female; Gene Expression; Genetic Vectors; Humans; Male; Megakaryocytes; Middle Aged; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad4 Protein; Thrombocythemia, Essential; Trans-Activators; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

Angiogenesis is critical for the clinical progression of haematopoietic malignancies and depends on angiogenic factors. Angiogenin is a powerful factor produced by neoplastic cells and host microenvironment. High levels of soluble angiogenin (sAng) correlate with a poor prognosis in patients affected by acute myeloid leukaemia and myelodysplastic syndromes, but no data are available on sAng in chronic myeloproliferative disorders (CMD). Therefore, in this study we investigated the clinical significance of the angiogenin in sera of patients with chronic myeloid leukaemia (CML) (n = 14) or essential thrombocythaemia (ET) (n = 20), and correlated them with those of soluble transforming growth factor-beta(1) (sTGF beta(1)). Enzyme-linked immunosorbent assay detected (P < 0.05) higher levels of sAng in CMD compared with healthy subjects (1026.74 +/- 464.60 pg/mL and 196.00 +/- 39.90 pg/mL, respectively). The highest levels of sAng were detected in CML patients (1349.23 +/- 549.55 pg/mL). Interestingly, CML patients who achieved haematological remission after interferon therapy showed circulating levels of angiogenin significantly (P < 0.05) decreased when compared with those at diagnosis. In ET patients, levels of angiogenin (889.34 +/- 267.66 pg/mL) and sTGF beta(1) (76.69 +/-6.08 pg/mL) were higher (P < 0.05) compared with healthy controls (57.93 +/- 19.39 pg/mL). No correlation was found between levels of sAng and levels of sTGF beta(1) or platelet count among ET patients. Our results show for the first time that elevated blood levels of angiogenin feature chronic myeloid malignancies, suggesting a role of angiogenin in the pathogenesis of these diseases.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Case-Control Studies; Chronic Disease; Female; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Myeloproliferative Disorders; Neoplasm Proteins; Prognosis; Remission Induction; Ribonuclease, Pancreatic; Solubility; Thrombocythemia, Essential; Transforming Growth Factor beta

Transforming growth factor-beta 1 (TGF-beta 1) is a cytokine which exhibits pleiotropic effects on many cell types and cellular systems. TGF-beta 1 has been shown to play a modulatory role in haematopoiesis and immunoregulation, expressed through its ability to inhibit the activities induced by other cytokines; however, the mechanisms underlying this activity are currently unclear. The potency of this activity varies according to the selected stimulatory cytokine and we have found that the proliferation of leukaemic cell lines induced by interleukin 4 (IL-4) is particularly sensitive to inhibition by TGF-beta 1 and provides a useful model to study the mechanism of action of TGF-beta. We have previously shown that IL-4 mediated mitogenic signal transduction in human systems involves the induction of phosphatase activity leading to the dephosphorylation of an 80-kDa protein (p80). We now show that TGF-beta 1 inhibits IL-4 induced dephosphorylation of p80 in a dose responsive manner closely correlated with its ability to inhibit the biological activity of IL-4. This suggests that TGF-beta 1 is inhibiting the same protein-tyrosine-phosphatase required by IL-4 to transduce its mitogenic signal. The biochemical mechanism underlying the biological activity of TGF-beta 1 in inhibiting IL-4 bioactivity is therefore the blocking of post receptor binding signal transduction processes.

Topics: Amino Acids; Cell Division; Humans; Interleukin-4; Leukemia, Erythroblastic, Acute; Phosphates; Phosphoproteins; Phosphorylation; Protein Tyrosine Phosphatases; Recombinant Proteins; Signal Transduction; Thrombocythemia, Essential; Transforming Growth Factor beta; Tumor Cells, Cultured

In this study we evaluated the amount of transforming growth factor-beta 1 (TGF-beta 1) in platelet lysates obtained from 12 patients affected by essential thrombocythaemia (ET) in comparison with five patients affected by myelofibrosis with myeloid metaplasia (MMM) and 15 healthy donors. The levels of both bioactive and latent TGF-beta 1, evaluated in a bioassay on CCL64 cells, before and after transient acidification, were similar in platelet lysates from ET patients and normal donors and significantly (P < 0.01) elevated in platelet lysates from MMM patients. Moreover, platelet lysates from ET patients and normal controls, showed a similar degree of colony suppression when tested on haematopoietic progenitor (CD34+) cells, purified from normal bone marrows, whereas platelet lysates from MMM patients showed a higher (P < 0.01) inhibitory activity on normal CFU-meg and BFU-E growth. In parallel, platelet lysates form ET patients and normal controls were tested on CD34+ cells, purified from ET bone marrows. ET bone marrow BFU-E, similarly to normal bone marrow BFU-E, were markedly inhibited by platelet lysates, whereas ET bone marrow CFU-meg were significantly (P < 0.05) less responsive to the inhibitory activity of platelet lysates than normal bone marrow CFU-meg. The main factor responsible for the inhibitory activity contained in platelet lysates was transforming growth factor-beta 1 (TGF-beta 1), as demonstrated by the ability of a polyclonal neutralizing anti-TGF-beta 1 antibody to almost completely reverse the suppressive effect of platelet lysates on CFU-meg and BFU-E growth. Our data demonstrate that the amount of intraplatelet TGF-beta 1 is similar in ET patients and normal controls, whereas it is increased in platelets from MMM patients. Moreover, megakaryocyte progenitors in ET show a reduced sensitivity to platelet-derived inhibitors and, in particular, to TGF-beta 1.

Topics: Blood Platelets; Cells, Cultured; Depression, Chemical; Dose-Response Relationship, Drug; Female; Hematopoietic Stem Cells; Humans; Male; Megakaryocytes; Middle Aged; Primary Myelofibrosis; Thrombocythemia, Essential; Transforming Growth Factor beta

Topics: Blood Platelets; Cells, Cultured; Colony-Forming Units Assay; Culture Media, Serum-Free; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Humans; Interleukin-3; Megakaryocytes; Thrombocythemia, Essential; Transforming Growth Factor beta

Topics: Bone Marrow; Cell Division; Cells, Cultured; Colony-Forming Units Assay; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Humans; Interleukin-3; Kinetics; Megakaryocytes; Recombinant Proteins; Thrombocythemia, Essential; Transforming Growth Factor beta