Megakaryocyte contribution to bone marrow fibrosis: many arrows in the quiver

In Primary Myelofibrosis (PMF), megakaryocyte dysplasia/hyperplasia determines the release of inflammatory cytokines that, in turn, stimulate stromal cells and induce bone marrow fibrosis. The pathogenic mechanism and the cells responsible for progression to bone marrow fibrosis in PMF are not completely understood. This review article aims to provide an overview of the crucial role of megakaryocytes in myelofibrosis by discussing the role and the altered secretion of megakaryocyte-derived soluble factors, enzymes and extracellular matrices that are known to induce bone marrow fibrosis. Additionally, we describe recent evidences showing that the role of megakaryocytes in tissue fibrosis is not limited to the bone marrow

DDR1 activation determines megakaryocyte migration on type I collagen.

Background. The mechanisms that regulate the megakaryocyte (MK) development within the bone marrow environment remain poorly understood. The underlying relationships between MK maturation and bone marrow components are key factors in this process. MK development occurs in a complex microenvironment where extracellular matrices are fundamental regulatory components. Since MK maturation is critical to blood functions, it appears evident that a fine regulation of the different steps of MK development is needed. On this basis, the search for new receptors, that may regulate MK functions, is still open. Discoidin domain receptor 1 (DDR1) is a tyrosine-kinase collagen receptor that is stimulated by fibrillar and base membrane collagens and mediates cell adhesion and migration in different tissues. Interestingly, DDR1 effects vary depending on cellular type and environment. Regarding the mechanisms, dissociation of actin from myosin IIa enhances DDR1 interaction with myosin IIa and thereby promotes cell migration. Further, DDR1 interacts with the non-receptor tyrosine-kinase Syk and the Syk-mediated cell migration inhibition is blocked in the presence of DDR1. Expression and function of DDR1 on human MKs are still completely unknown. Hypothesis. The hypothesis for the present work is that DDR1 may represent a new collagen receptor of human MKs that regulates their functions in the bone marrow-matrix environment. Methods. MKs were differentiated from human cord blood hemopoietic progenitors. DDR1 expression in mature MKs was evaluated by RT-PCR, immunofluorescence and western blotting analysis. DDR1 activation and interaction with other molecules was evaluated by immunoprecipitation. For cell migration experiments, MKs were seeded in the upper well of transwell migration chambers. MK migration, towards SDF-1 and through filters coated with type I collagen containing or not a DDR1 blocking molecule (DDR1:Fc), was measured after 16 hours. To study the phosphorylation status of DDR1 downstream molecules, western blotting analysis was performed. Results. Our results showed that human MKs express DDR1 at both mRNA and protein levels. MK DDR1 was activated by incubation with type I collagen as demonstrated by immunoprecipitation and probing with an anti-phosphotyrosine antibody. Moreover, upon type I collagen stimulation, MK DDR1 associated with myosin IIA as demonstrated by co-immunoprecipitation. In order to study the role of DDR1 in mediating MK interactions with type I collagen, we took advantage of the recombinant soluble protein DDR1:Fc that was previously shown to block DDR1 activation. Immunoprecipitation analysis demonstrated that DDR1 activation was inhibited in MKs that were let to adhere for 16 hours on type I collagen treated with DDR1:Fc as compared to control. Interestingly, no differences in MLC-2 phosphorylation were observed in MKs adherent on either type I collagen preparations. Consistently, DDR1:Fc did not affect neither MK adhesion nor MK spreading on type I collagen demonstrating that the engagement of other collagen receptor, such as alpha2beta1 integrin, had occurred. However, an important increase of Src and Syk non receptor kinase activation was observed in MKs upon adhesion on DDR1:Fc treated type I collagen with respect to controls. Importantly, a significant decrease (about 40%) of MK migration, through type I collagen, was observed when DDR1 was blocked as compared to controls. These results demonstrated that Syk-mediated migration inhibition is blocked by activation of DDR1 in human MKs. Conclusions. In conclusion, this study provides evidence that the new collagen receptor DDR1 may participate in the regulation of human MK interactions and functions on type I collagen. Although preliminary, these data demonstrate that the bone marrow-matrix environment regulate MK functions through multi-faceted mechanisms that are still to be completely unraveled

A newly identified platelet and megakaryocyte lysyl oxidase adhesion to collagen axis in human primary myelofibrosis

Controlling platelet function is central to management of various pathologies, including Primary Myelofibrosis (PMF), which is associated with increased incidence of thrombosis and cardiovascular disease. In recent studies we showed that the matrix cross-linking enzyme, Lysyl Oxidase (LOX) is elevated in platelets and megakartocytes of myelofibrotic mice, and transgenic upregulation of LOX increases platelet and megakaryocyte adhesion to monomeric type I collagen (preferred by alpha2β1 collagen receptors), and augments propensity for in vivo thrombosis. Here, we examined the relevance of these findings to human disease, by first determining platelet LOX level, as well as platelet and megakaryocyte adhesion to collagen using samples derived from PMF patients and matching controls. In analyzing 10 PMF platelet samples (5 males and 5 females; 6 JAK2V617F; 4 CALR mutations; age range 30-55; PMF grade 1-3), we found a nearly 20 fold upregulation of LOX expression compared to matching healthy controls (p<0.001). Intriguingly, there was a significant increase in adhesion (plt/mm2) and spreading (pixel2) of PMF platelets relative to control on monomeric, pepsinated acid soluble collagen (PSCI) (p<0.05), while no differences were observed between the samples on native triple helical acid soluble collagen type I collagen (ASCI). To examine the role of LOX in this phenotype, we treated control and PMF-derived human megakaryocytes, differentiated from peripheral blood CD34+ cells, grown in presence or not of LOX inhibitor, β-aminopropionitrile (BAPN) from day 2 of culture. Our preliminary data, based on a cohort of 2 controls and 5 PMF samples, demonstrated that although on ASCI megakaryocyte adhesion is not altered by BAPN treatment both in CTRL and PMF derived megakaryocytes, on PSCI the adhesion of PMF derived megakaryocytes was reduced by about a 50% by BAPN treatment, while the adhesion of CTRL derived MKs was not significantly affected. Taken together, we identified LOX level to be upregulated in human PMF platelets and megakaryocytes, and LOX activity to be important for PMF cells adhesion to collagen. These newly identified properties are highly relevant to megakaryocyte adhesion to the niche, and to platelet activation in PMF

Mechanisms of platelet release: in vivo studies and in vitro modeling

Mechanisms related to platelet release in the context of the bone marrow niche are not completely known. In this review we discuss what has been discovered about four critical aspects of this process: 1) the bone marrow niche organization, 2) the role of the extracellular matrix components, 3) the mechanisms by which megakaryocytes release platelets and 4) the novel approaches to mimic the bone marrow environment and produce platelets ex vivo

Constitutive STAT5 phosphorylation in CD34+ cells of patients with primary myelofibrosis: Correlation with driver mutation status and disease severity.

Primary Myelofibrosis (PMF) is a myeloproliferative disorder associated with JAK2V617F, Calreticulin (CALR) indels, and MPLW515L/K mutations activating the tyrosine kinase JAK2 and its downstream signaling pathway. The nature of signaling abnormalities in primary cells from PMF patients is poorly understood, since most of the work has been performed in cell lines or animal models. By flow cytometry we measured constitutive and cytokine induced phosphorylation of STAT5, STAT3, and ERK1/2 in circulating CD34+ cells from 57 patients with PMF (20 with prefibrotic-PMF) and 13 healthy controls (CTRLs). Levels of constitutive and TPO induced p-STAT5, and IL6 induced p-STAT3 were higher in patients than in CTRLs. Constitutive p-STAT5 values were lower in CALR than homozygous JAK2V617F mutated CD34+ cells from PMF patients. Moreover, constitutive p-STAT5 and IL6 induced p-STAT3 values correlated directly with circulating CD34+ cell number/L, and inversely with the frequency of circulating CD34+ cells expressing CXCR4. Constitutive p-STAT5 values of CD34+ cells were also inversely correlated with hemoglobin levels. When the patients were divided according with presence/absence of JAK2V617F mutation, all the correlations described characterized the JAK2V617F+ patients with prefibrotic-PMF (P-PMF). In conclusion, increased constitutive p-STAT5 and IL6 induced p-STAT3 values in circulating CD34+ cells characterize patients with PMF. Constitutive p-STAT5 and IL6 induced p-STAT3 values correlate with circulating CD34+ cell number/L, the frequency of circulating CD34+ cells expressing CXCR4 and hemoglobin levels within the prefibrotic JAK2V617F+ patient population. Our data point toward a complex activation of STAT5-dependent pathways in the stem/progenitor cell compartment, that characterize the phenotypic diversity of PMF