Myelofibrosis and osteosclerosis are prominent features arising in mice overexpressing thrombopoietin (TPO). The pivotal role of transforming growth factor beta 1 (TGF-beta 1) in the pathogenesis of myelofibrosis has been documented, but the mechanisms mediating osteosclerosis remain unclear. Here, we used mice deficient in osteoprotegerin (OPG), a secreted inhibitor of bone resorption, to determine whether osteosclerosis occurs through a deregulation of osteoclastogenesis. Marrow cells from opg-deficient mice (opg(-/-)) or wild-type (WT) littermates were infected with a retrovirus encoding TPO and engrafted into an opg(-/-) or WT background for long-term reconstitution. The 4 combinations of graft/host (WT/WT, opg(-/-)/opg(-/-), opg(-/-)/WT, and WT/opg(-/-)) were studied. Elevation of TPO and TGF-beta 1 levels in plasma was similar in the 4 experimental groups and all the mice developed a similar myeloproliferative syndrome associated with severe myelofibrosis. Osteosclerosis developed in WT hosts engrafted with WT or opg(-/-) hematopoietic cells and was associated with increased OPG levels in plasma and decreased osteoclastogenesis. In contrast, opg(-/-) hosts exhibited an osteoporotic phenotype and a growth of bone trabeculae was rarely seen. These findings suggest that osteosclerosis in mice with TPO overexpression occurs predominantly via an up-regulation of OPG in host stromal cells leading to disruption of osteoclastogenesis.

Objective. Osteoprotegerin (OPG) is a soluble member of the tumor necrosis factor receptor superfamily critically involved in the regulation of bone resorption. Within the bone microenvironment, OPG is abundantly produced by osteoblast/stromal cells, and its expression is regulated by transforming growth factor-beta(1) (TGF-beta(1)). However, OPG expression and regulation in primary hematopoietic cells have not been fully investigated.Materials and Methods. Opg mRNA was studied in murine hematopoietic cells by semiquantitative reverse transcriptase-polymerase chain reaction. The OPG protein was identified by immunofluorescence labeling and secretion was assessed by enzyme-linked immunosorbent assay.Results. Opg transcripts were detected in platelets, megakaryocytes (MK), monocytes, and B lymphocytes, but not in erythroblasts, neutrophils, and T lymphocytes. Mature MK and proplatelets exhibited strong immunostaining for OPG outside the storage alpha-granules, and secretion was detected in the conditioned medium. To analyze whether opg transcription in MK was influenced by TGF-beta(1), the opg/GpIIb mRNA ratio was compared in cultured MK derived from TGF-beta(1) null mutants and wild-type littermates without or after the addition of bioactive TGF-beta(1). No difference was seen, indicating that opg expression in MK was not modulated by TGF-beta(1). However, mRNA levels were increased when thrombopoietin was present in the culture medium, suggesting that MK maturation was correlated with enhanced opg expression.Conclusions. With these results we document for the first time that murine MK and platelets express OPG. This suggests a novel role for MK in bone homeostasis, in addition to its role in vascular homeostasis. (C) 2003 International Society for Experimental Hematology. Published by Elsevier Inc.

In TGF-beta1-knockout mice, TGF-beta1-null conceptuses die during embryonic development with a penetrance of lethality that depends on the mouse genetic background. Studies have suggested that transplacental passage of maternal TGF-beta1 could account for the rescue of some TGF-beta1-null embryos. Herein, we have used an adenovirus-based gene delivery system and a strain of mice where most TGF-beta1-null conceptuses die prior to parturition, to investigate whether an increase in maternal TGF-beta1 during pregnancy would rescue TGF-beta1-null embryos. A single intravenous injection of an adenovirus containing a modified version of TGF-beta1 cDNA (Ad-TGF-beta1S223/S225), coding for a biologically active form of the cytokine, induced a 20-fold increase in plasma TGF-beta1 (active and latent forms) levels for up to 3 months in adult mice. Similar levels of TGF-beta1 were detected in 13-day post coïtum (dpc) embryos from Ad-TGF-beta1-treated mothers, demonstrating an efficient maternal/fetal transfer of the cytokine. However, no increase in the frequencies of TGF-beta1-null neonates nor in day 11.5 dpc TGF-beta1-null conceptuses was observed despite elevated levels of TGF-beta1 delivered throughout gestation. In addition, we show that the high levels of TGF-beta1-titrated in the plasma from Ad-TGF-beta1S223/S225-treated mice were partly the consequence of a stimulation of an autocrine production by exogenous bioactive TGF-beta1. These results indicate that transplacental passage of TGF-beta1 was not effective in rescuing TGF-beta1-null conceptuses from embryonic lethality.

The cyclin-dependent kinase inhibitor p21(Waf-1/Cip-1) is expressed at high level during megakaryocyte differentiation, but its precise function remains unknown. In this study, it is confirmed that p21 was expressed at a high level in hypoploid (2N and 4N) and polyploid (at least 8N) human megakaryocytes derived from CD34(+) cells. A high expression of p27(Kip1), p16, cyclin E, and cyclin D3 was also found in both populations associated with a hypophosphorylated form of retinoblastoma protein, suggesting that the majority of hypoploid and polyploid megakaryocytes are G(1)-arrested cells. As human megakaryocytes grown in vitro present a defect in their polyploidization, the study switched to the murine model. The modal ploidy of megakaryocytes derived from lineage-negative cells was 32N, and an elevated expression of p21 was found in high-ploidy megakaryocytes. In addition, p21 and p27 were coexpressed in the majority of mature polyploid megakaryocytes. The p21 was detected by immunofluorescence in megakaryocytes derived from p53(-/-) mice, demonstrating a p53-independent regulation during megakaryocyte differentiation. Megakaryocytopoiesis of p21(-/-) mice was subsequently studied. No marked abnormality in the ploidy of primary or cultured megakaryocytes was detected. Overexpression of p21 in p21(-/-) or normal murine megakaryocytes and in human megakaryocytes showed in all these cases a marked inhibition in megakaryocyte polyploidization. In conclusion, while a reciprocal relation is observed between p21 levels in megakaryocytes and the cycling state of the cells, p21 is not essential for the determination of the ploidy profile in normal megakaryocytes in vivo. However, high levels of its expression in cultured megakaryocytes arrest the endomitotic cell cycle.

The development of chronic myelogenous leukemia (CML) models in mice using an inducible BCR-ABL gene has been hampered by the requirement of sequential expression of tTA (Tet repressor-VP16 fusion protein) and Tet-OP sequences in the same cells after separate transfection. This double transfection strategy is time consuming as it requires screening of many hundreds of individual clones and cannot be applied to primary hematopoietic cells. To generate a tetracycline-inducible BCR-ABL retrovirus, we have subcloned BCR-ABL p210 cDNA in the SIN-Retro-TET vector, which allows regulated expression of a gene of interest in a single autoregulatory cassette, containing both tTA and Tet OP sequences. Retroviral particles were obtained by transfecting the SIN-BCR-ABL p210 construct into the 293 cells and by VSVG pseudotyping. To determine the functionality of the retrovirus, the IL-3-dependent murine Ba/F3 cell line was retrovirally transduced and clones were grown in the absence of both IL-3 (to select for transformed cells) and a tetracycline analog, doxycycline (to induce BCR-ABL expression). Using this technique, polyclonal Ba/F3 cells and several growth factor-independent Ba/F3 clones expressing BCR-ABL were obtained within 2–3 weeks. A single dose of doxycycline added to the medium (1 μg/ml), induced in different clones, a reduction of BCR-ABL protein levels by 60–90% at 24 h, leading to cell death in the absence of IL-3. In several individual clones, BCR-ABL expression was further reduced to become almost undetectable at 48 h. The doxycycline-regulated BCR-ABL expression was stable, as many clones maintained in culture for >8 months showed a persistent inhibitory response to doxycycline addition in the medium. In in vivo experiments, subcutaneous injection of 2 × 106 Ba/F3-SIN p210 cells in nude mice induced visible tumors in 2 weeks and all established tumors completely regressed upon addition of doxycycline in the drinking water (200 μg/ml). To determine the functionality of the inducible BCR-ABL retrovirus in vivo, primary Lin− bone marrow cells were transduced with SIN-p210 and transplanted in lethally irradiated mice. All transplanted mice had successful hematopoietic reconstitution and BCR-ABL integration was found in the peripheral blood of seven out of 14 mice available for long-term analysis (>6 months). However, despite evidence of retrovirus-mediated gene transfer, there was no evidence of leukemia, due either to low viral titers or to the relative inefficiency of the minimal CMV promoter in primary hematopoietic cells. Thus, these results demonstrate for the first time, to our knowledge, the feasibility to generate an inducible BCR-ABL retrovirus in a single step, in the context of an immortalized cell line. Our data suggest that with further improvements of the retrovirus-mediated gene transfer technology, it might be possible to generate inducible leukemia models in mice by the use of single retroviral constructs.

The thrombocytopenia and absent radii (TAR) syndrome is a rare disease associating bilateral radial agenesis and congenital thrombocytopenia. Here, we investigated in vitro megakaryocyte (MK) differentiation and expression of c-mpl in 6 patients. Using blood or marrow CD34(+) cells, the colony-forming unit (CFU)-MK number was markedly reduced. CD34(+) cells were also cultured in liquid medium in the presence of a combination of 3 cytokines (stem cell factor, interleukin-3, and interleukin-6) or megakaryocyte growth and development factor (PEG-rHuMGDF) with or without SCF. In the presence of PEG-rHuMGDF, the majority of mature megakaryocytes (CD41 high, CD42 high) underwent apoptosis. This phenomenon was also observed in cultures stimulated by three cytokines. However, this last combination of cytokines allowed a more complete terminal MK differentiation. Surprisingly, a homogeneous population of CD34(-)CD41(+)CD42(-) cells accumulated during the cultures. This population was unable to differentiate along the myeloid pathways. This result suggests that a fraction of MK cells is unable to differentiate in the TAR syndrome. We subsequently investigated whether this could be related to an abnormality in c-mpl. No mutation or rearrangement in the c-mpl gene was found by Southern blots or by sequencing of the c-mpl coding region and its promoter in any of the patients. Using Western blot analysis, a decreased level of Mpl was found in patient platelets. A decreased level of c-mpl messenger RNA in TAR platelets was also detected with a lower c-mpl-P to c-mpl-K ratio in comparison to adult platelets. Altogether, these results demonstrate that the thrombocytopenia of the TAR syndrome is associated with a dysmegakaryocytopoiesis characterized by cells blocked at an early stage of differentiation. (Blood. 2000;95:1633-1641)

Liver synthesizes thrombopoietin, which is a major cytokine involved in the production of hematopoietic cells. The purpose of this study was to examine the effects of preferential liver irradiation on expression of thrombopoietin and production of hematopoietic cells. About 70% of the liver of C57BL6/J mice was irradiated with 20 Gy of gamma rays. Exposure to ionizing radiation enhanced hematopoietic progenitors and megakaryocyte frequency in bone marrow and induced a transient increase in platelet and neutrophil counts that peaked 14 days after irradiation. The concentration of thrombopoietin was increased in serum as early as 5 h after liver irradiation and was still elevated at day 14. By using Northern blot analysis and an RNase protection assay, we showed that thrombopoietin mRNA was increased in the irradiated liver. To determine whether thrombopoietin was involved in the stimulation of hematopoiesis, we irradiated mice in which thrombopoietin deficiency had been induced by homologous recombination. Platelet levels were increased in both heterozygous and homozygous thrombopoietin-deficient mice with a magnitude similar to that obtained in normal mice. In summary, our data demonstrate that local irradiation of the abdomen encompassing the liver leads to stimulation of hematopoiesis through a thrombopoietin-independent mechanism.

BACKGROUND AND OBJECTIVE:Thrombopoietin (TPO), also referred to as MpI ligand, is the most potent cytokine that physiologically regulates platelet production. With the availability of sufficient amounts of recombinant forms of the protein, the biological in vitro and in vivo activities of this cytokine have been extensively studied. The objective of this review is to summarize the published data focusing on TPO production and regulation and to discuss the pleiotropic biological action of this hormone. The review also highlights the results so far obtained in preclinical and clinical trials. EVIDENCE AND INFORMATION SOURCES:The material examined in this review includes data published by the author and articles or abstracts published in Journals covered by Medline. The author has contributed to the isolation of TPO, has been working in the field for several years and has contributed original papers on the TPO/MpI system in normal and pathologic situations. STATE OF THE ART:TPO is a hormone constitutively produced by the liver and kidneys. Plasma levels of TPO are regulated through receptor-mediated uptake, internalization and catabolism. First thought to be a lineage dominant factor promoting megakaryocytopoiesis, several lines of evidence indicate that TPO has pleiotropic effects on hematopoiesis. In vitro studies show that TPO alone, or in combination with early acting cytokines, stimulates the proliferation and enhances the expansion of primitive CD34+ CD38- hematopoietic progenitor cells. In vivo studies with c-mpl- and TPO-null mice reveal that the molecule sustains the survival and proliferation of early committed progenitor cells of various type. Preclinical and clinical trials indicate that recombinant TPO molecules increase platelet counts and megakaryocyte numbers in normal or mildly thrombocytopenic states. However, no significant effects of TPO administration on platelet recovery have so far been reported in patients subjected to intensive chemotherapy regimens. Recombinant molecules appear to be safe to administer and very little toxicity is reported. TPO augments the number of erythroid and myeloid committed progenitor cells in marrow, and mobilized stem cells in peripheral blood. PERSPECTIVES:The potential clinical use of TPO is still unclear. With the increased knowledge of the multiple effects of TPO on hematopoiesis, it is expected that future carefully monitored clinical trials will provide more information regarding the eventual benefits of this cytokine in the treatment of thrombocytopenia. At present, one successful application of TPO appears to be its addition in cytokine cocktails used to expand hematopoietic stem cells ex vivo.

The discovery of the Mpl ligand (Mpl-L), also called thrombopoietin (TPO), has facilitated in vitro investigation of human megakaryocytopoiesis. By confocal microscopy, endomitosis appeared as abortive mitosis skipping late stages of mitosis. No telophase and cytokinesis were observed. A spherical multipolar spindle which limits chromatid segregation was observed. The nuclear envelope subsequently reformed isolating all chromatids in a single nucleus. Platelet shedding was ultrastructurally studied. Platelet release occurred after formation of long cytoplasmic extensions (proplatelet formation), constriction areas delineating platelet territories. Heterogeneity in platelet size may be determined by the length of these extensions. Pegylated-recombinant human megakaryocyte growth and development factor, a truncated form of Mpl-L, was the most efficient cytokine to produce proplatelet-bearing megakaryocytes (MKs) and platelets in vitro. However, functional platelets with a normal ultrastructure could be produced in the presence of a combination of other cytokines. Finally, we investigated whether the induction of MK differentiation by the MS-5 stromal cell lines is due to Mpl-L. MS-5 cells synthesized Mpl-L transcripts and a biologically active protein. When human CD34+ cells were grown in contact or noncontact cultures with MS-5 cells, MK differentiation was observed. Soluble Mpl receptor (sMpl-Fc) addition inhibited MK growth, suggesting that the MK-promoting activity was due to Mpl-L production. Marrow stromal cell lines derived from TPO-/- mice were also able to sustain MK growth. Despite the absence of any production of Mpl-L, the sMpl-Fc continued to inhibit MK differentiation. This result suggests that the sMpl has a direct inhibitory effect and may explain the divergent results in the literature concerning the precise role of Mpl-L on the MK terminal differentiation.

Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.

Thrombopoietin (TPO), the primary physiological regulator of platelet production, was initially thought to be a lineage-specific factor acting predominantly on megakaryocytopoiesis. Detailed studies establish that this cytokine mediates biological effects on a broad spectrum of hematopoietic progenitor cells, including stem cells. TPO is a hormone constitutively produced mainly by the liver and kidney. Plasma TPO levels are regulated by the platelet and megakaryocyte mass through Mpl receptor binding, internalization and degradation. The Mpl receptor is a member of the hematopoietin receptor superfamily lacking intrinsic kinase activity, Upon ligand-induced Mpl homodimerization, the major signaling events for proliferation are mediated through the JAK2/STAT5 pathway, while differentiation might occur through a prolonged activation of the MAPK pathway. Preclinical and clinical studies demonstrate the potential use of TPO in a variety of contexts, but it is too early to evaluate its benefit in reducing platelet transfusion.

Regulation of megakaryocytopoiesis and platelet production is a complex phenomenon. It has been demonstrated that numerous pleiotropic cytokines act in vivo and in vitro on megakaryocytopoiesis. Historically, studies on the regulation of megakaryocytopoiesis were largely dominated by the concept of humoral regulation. Despite 35 years of work, the factor responsible for these effects (thrombopoietin, TPO) could not be purified to homogeneity. The proto-oncogene c-mpl has been demonstrated to encode a protein which is the receptor for a humoral cytokine regulating megakaryocytopoiesis. Its ligand was isolated by three independent groups. The purified protein was termed Mpl-L (Mpl-Ligand), thrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF). Strong evidence that Mpl-L is the homeostatic regulator of platelet production has been provided by c-mpl or Mpl-L knock-out mice which have a severe but not lethal thrombocytopenia. In vitro experiments have indicated that the Mpl-L acts both as a proliferative and differentiative factor as many other CSF. At the unicellular level, Mpl-L acts on MK progenitors inducing their proliferation. Mpl-L induces polyploidisation of the MKs and cytoplasmic maturation leading to an in vitro platelet production. As a single cytokine Mpl-L is the most potent growth factor for the MK lineage in vitro. However, a combination of cytokines can totally replace the effects of Mpl-L both on proliferation of MK progenitors and their maturation. In addition, Mpl-L has a major effect on primitive hematopoietic progenitors.

Thrombopoietin (TPO) is the central regulator of megakaryocytopoiesis and thrombocytopoiesis. Preclinical data and human studies have so far shown that the recombinant molecule is safe to administer and associated with very little toxicity. Nevertheless, different experimental animal models have revealed that a chronic exposure to very high doses of TPO could result in myeloproliferative syndromes with a spectrum of pathological features in common with human idiopathic myelofibrosis (PMF). A number of investigators have researched whether TPO or its receptor Mpl were involved in the pathogenesis of human myeloproliferative syndromes which are also characterized by a predominant megakaryocytic involvement, in PMF and primitive essential thrombocythemia. In both diseases, megakaryocyte (MK) progenitors develop autonomously in serum-deprived cultures. This spontaneous MK development is also observed at limiting dilution demonstrating that MK escape the normal regulatory controls. Furthermore, this abnormal MK proliferation and maturation is neither due to an autocrine stimulation by TPO nor by point mutation or deletion in the coding region of the c-mpl gene. This paper will review the data that have been reported to date on the effects of an overexpression of Mpl ligand and related molecules on the induction of experimental myelofibrosis and highlight recent insights into the pathogenesis of PMF.

Adenoviral vectors may be useful tools to deliver a cytokine in vivo. A single intravenous injection of an adenovirus vector containing the human thrombopoietin (TPO) cDNA (AdRSVhuTPO) was able to induce a thrombocytosis for more than 6 weeks in SCID mice, associated with a megakaryocyte (MK) hyperplasia in different organs. A marrow and spleen fibrosis was observed at 6 weeks. In immunocompetent mice, a single AdRSVhuTPO injection led to a moderate and transient thrombocytosis without myelofibrosis. To evaluate the usefulness of TPO for the prevention of secondary side-effects during an aplastic period, mice were subjected to a myeloablative regimen 7 days after the intravenous AdRSVhuTPO injection. In this setting, TPO prevented mortality by accelerating hematological recovery. Survival was essentially related to an improvement in the leukopenia since all control mice died from septicemia. However, the effects of TPO may be potentiated by the release of inflammatory cytokines following the adenovirus infection; AdRSVβgalactosidase injected-mice had higher numbers of BFU-E and CFU-GM in the marrow than PBS-injected mice. Myelosuppression induced transient immunosuppression responsible for a sustained expression and elevation of platelet numbers for at least 5 months. These results further suggest that TPO may be an effective therapy in diminishing hematological complications related to myeloablative regimens, but emphasize that immunosuppression secondary to myelosuppression may lead to sustained expression associated with a risk of thrombosis and myelofibrosis when delivered by adenovirus vectors.

To evaluate the effects of long-term high-dose exposure to Mpl-ligand also called thrombopoietin (TPO), C57BL/6J murine marrow cells were infected with a retrovirus carrying the murine TPO gene. Mice were treated 4 days by 5-FU and marrow cells were then infected by coculture using a MPZen vector containing the murine TPO cDNA. Non adherent marrow cells were transplanted into lethally irradiated recipients. A majority of hematopoietic cells in the marrow, spleen, thymus and blood was transduced by the retroviral vector, one and three months after reconstitution. Plasma TPO activity in transplanted mice was extremely high (10 4 U/ml). A disease with two distinct steps was observed. During the two first months after transplantation, platelet (plt) and white blood cell (WBC) counts increased 4- and 10-fold, respectively. Abnormal platelet size and granules were observed. Spleen weight increased 4-fold and marrow cellularity decreased 5-fold. Histology revealed hyperplasia of the megakaryocytic and myeloid cells. Total numbers of CFU-MK and CFU-GM increased. In contrast, the hematocrit progressively fell accompanied by a decrease in the erythroblasts and CFU-E numbers. Beginning two months after transplantation, plt and WBC numbers also declined. Thrombocytopenia was noted 5 months after transplantation. The Hcts continued to decrease. Few cells were isolated from the marrow cavities and spleens. Histology revealed fibrosis of the marrow and spleen and significant osteosclerosis of the marrow. An extramedullary hematopoiesis was observed in numerous organs such as the liver or the kidney. Total numbers of progenitors were very low in hematopoietic organs. Mice died 7 months after transplantation with severe pancytopenia. Two early deaths were observed with a marked increase in blast cells. This disorder was transplantable into secondary recipients who developed an attenuated form of the disease similar to the one previously described [Yan et al (1995) Blood 86: 4025]. In conclusion, dysregulated TPO production by hemopoietic cells in mice results in a fatal myeloproliferative disease which mimics the clinical evolution of idiopathic myelofibrosis observed in man.