Bone Marrow CFU-MK Research Articles

Imatinib mesylate has an inhibitory effect on hematopoiesis in a mice model

Imatinib mesylate (Gleevec or STI-571), a tyrosine kinase inhibitor, can potently block both platelet-derived growth factor (PDGF) receptor-α and β by inhibiting the receptor tyrosine kinase activity. Imatinib mesylate has been successfully used to treat patients with chronic myeloid leukemia (CML) and Ph positive acute lymphoid leukemia (ALL). However, a significant number of patients with imatinib mesylate treatment have developed neutropenia, thrombocytopenia and anemia (van Deventer, Hall et al. 2002). Our previous studies have demonstrated that PDGF/PDGFR plays an important role on thrombopoiesis, and blockage of PDGFR by imatinib inhibited the downstream signal pathway, which led to reduce of megakaryocyte production (Ye, et al. Haematologica, 2010). This may partially explain the imatinib-related thrombocytopenia. However, the role of imatinib on normal hematopoiesis remains to be investigated. In the present study, we identified the expression of PDGFR-β on the human megakaryoblastic cells: Meg-01, CHRF-288-11, Dami and M-07e, human erythromyeloblastoid leukemia cells: K-562 and human myeloblastic cells: HL-60 by RT-PCR, western blot and flow cytometry. We then demonstrated the effect of imatinib on normal hematopoiesis in the 8 wk old C57BL/6 mice model. Gleevec (25 mg/kg/day) dissolved in distilled water or water only were given orally for up to 2 wks. 15ul whole blood was taken from postorbital weekly to perform blood cell counts. The Gleevec -treated group showed a reduction of platelet count as compared to the control at day 14 (543.3±71.95×109/L vs. 330.0±62.23×109/L, n=5, P<0.05). Similarly, Gleevec caused a significant decrease in the number of white blood cells on day 14 (9.667×±0.8511×109/L vs. 6.080±0.8015×109/L, n=5, P<0.05). While no significant differences were found on red blood cell count between Gleevec and control groups. The mice bone marrow was harvested from each group to perform CFU assays. Gleevec significantly inhibited the formation of bone marrow CFU-MK (15.33±1.764 vs 3.333±0.6667, n=3), and CFU-GM(13.50±2.784 vs 6.333±0.7265, n=3) compared with the control. There were no significant differences found in CFU-GEMM and CFU/BFU-E formation between the Gleevec and control treatment. Our studies suggested that PDGFR were expressed on hematopoietic cells. Imatinib mesylate, an inhibitor of PDGFR, not only played an important role in malignant hematopoiesis, but also in normal hematopoiesis. Administration of Gleevec reduced white blood cells and megakaryocytes/platelets production, which implied its potential therapeutic role in treating myeloproliferative diseases, such as Essential Thrombocytosis. Disclosures:No relevant conflicts of interest to declare.

RAD001-mediated STAT3 upregulation and megakaryocytic differentiation

RAD001 is currently used as an immunosuppressant and anticancer drug. Megakaryocyte (MK) differentiation includes development from pluripotent stem cells to proliferation and differentiation toward MK formation and platelet maturation. Our preliminary assay showed that RAD001 might stimulate MK differentiation; however, the exact regulatory mechanisms needed to be elucidated. By the ex vivo assay, RAD001 induced MK differentiation in human haematopoietic stem cells, with both the stimulation of CFU-GM colony formation and CD61 surface marker expression. Then, BALB/c mice were orally administrated with or without agrylin and/or RAD001 for 15 days. The platelet count and bone marrow CFU-MK colony formation were eliminated by agrylin, but unchanged in RAD001 and RAD001 plus agrylin mice. An ex vivo assay of bone marrow-derived stem cells demonstrated that RAD001 increased the number of CFU-MK colonies. The MK count in bone section indicated the decreased effect by agrylin and then recovered by RAD001. The level of plasma thrombopoietin was also enhanced in RAD001-treated mice. The effect of RAD001 on human leukaemic K562 and HEL cells showed the growth inhibition and MK differentiation activities; including morphological observation, CD41 and CD61 expression, and platelet factor 4 secretion. In RAD001-treated HEL cells, p-STAT3 expression, STAT3 translocation, and STAT3-DNA binding activity were up-regulated. Furthermore, STAT3 siRNA decreased the p-STAT3 and CD61 expression, as well as the CD61 fluorescence intensity, indicating that STAT3 may be critical in RAD001-mediated MK differentiation. Conclusion, the present study demonstrated that RAD001 might have the capacity to induce MK differentiation through the up-regulation of STAT3 signalling.

Angelica Polysaccharides Promotes Thrombopoiesis through the Phosphatidylinositol 3-Kinase/Akt Pathway.

Angelica Polysaccharide is from the root of Radix Angelicae Sinensis (Chinese Danggui). Danggui has been used for centuries to treat blood-deficiency related diseases. The hematopoietic effect of Danggui may be related to its constituent, polysaccharide. However, the effects of angelica polysaccharide on hematopoietic stem/progenitor cells and megakaryocyte/platelet lineage have not been well studied. In this study, we specifically investigate the thrombopoietic effect of Angelica polysaccharide (APS) in a mouse model and its molecular mechanism. A myelosuppression mouse model (4-Gy-irradiated) was treated with APS (10 mg/kg/day) and thrombopoietin (TPO) (1 mg/kg/day). Peripheral blood cells from APS, TPO and vehicle-treated samples were counted on days 0, 7, 14 and 21. Then CFU assays were used to determine the effects of APS on the megakaryocytic progenitor cells and other lineages. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were conducted in megakaryocytic cell line M-07e. The effects of APS on cells treated with Ly294002, a Phosphatidylinositol 3-Kinse inhibitor and the effect of APS on the phosphorylation of AKT were also studied. APS as well as TPO significantly enhanced the recovery of platelet and WBC count, and bone marrow CFU-MK and CFU-GM formation (n=6). Morphological examination of bone marrows showed that APS treatment significantly increased the recovery of the megakaryocytic series. APS also increased the recovery of the body weight and organ weight (liver and spleen). However, APS didn't have a up-regulating effect on TPO production by a ELISA assay. We further analyzed the in vitro effect of APS on CFU-MK and CFU-GM formation. APS (50 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK (p=0.06, n=4), and CFU-GM formation (p=0.032, n=6). However, APS alone (00 ug/ml) did not show significant effect on CFU-MK and CFU-GM proliferation (n=6). The effect of APS (1–500 ug/ml) on the growth of bone marrow stromal cells was further investigated using CFU-F (fibroblastoid) assay. APS (50 ug/ml) alone showed a promoting effect on CFU-F growth (p=0.049, n=3). APS (50 ug/ml) also significantly enhanced PDGF, bFGF and VEGF-induced CFU-F formation (n=4). Moreover, the anti-apoptotic effect of APS in M-07e cells was also demonstrated by using Annexin-V, Caspase-3, and JC-1 assays. Addition of Ly294002 alone increased the percentage of cells undergoing apoptosis. However, additional of APS to Ly294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the phosphorylation of AKT. APS promotes thrombopoiesis in a mouse model. This effect is likely to be mediated by the PI3K/AKT pathway.

VPAC1 Receptor-Mediated Regulation of Megakaryopoiesis.

Identification of the regulatory pathways that direct megakaryopoiesis and platelet production is essential for the development of novel strategies to treat life threatening bleeding complications in bone marrow suppressed patients. We demonstrated that megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which both PACAP and VIP are specific agonists. We have further identified a bleeding tendency and found three copies of the PACAP gene in two related patients with severe mental retardation, responsible for elevated PACAP plasma levels and associated increased platelet cAMP concentrations, resulting in strongly reduced platelet aggregation (JCI, 2004, 113, 905). In this study, we have further demonstrated a fundamental role for the VPAC1 signalling pathway during megakaryocyte maturation and platelet formation. Patients with PACAP overexpression have mild thrombocytopenia, a normal platelet survival and relatively small platelets with an MPV of 8.2 fL (normal MPV 9-13 fL). FACS analysis of the patients' platelets showed reduced expression of GPIX and GPIIIa. Electron microscopy of bone marrow of patients and of mice, specifically overexpressing PACAP in megakaryocytes revealed the presence of early megakaryocyte progenitors but almost not of mature megakaryocytes. Immature megakaryoblasts seemed to have reduced levels of rough endoplasmic reticulum cisternae and free ribosomes. To further study the modulating role of VPAC1 in thrombopoiesis, control mice were therefore subcutaneously injected with neutralizing polyclonal or monoclonal anti-PACAP, anti-VIP or anti-VPAC1 antibodies. Injection of these antibodies in all cases led to increased platelet counts, compared to control antibodies (monoclonal anti-PACAP antibody: 1194 ± 237 x 109 plt/L; control antibody: 722 ± 178 x 109 plt/L; p=0.01, unpaired t-test at day 7 after injection). This strategy was also capable of reducing the drop in platelet count in busulfan treated mice (polyclonal anti-PACAP antibody: 561 ± 121 x 109 plt/L; control antibody: 349 ± 65 x 109 plt/L; p=0.033, unpaired t-test at day 29 or day 18 after respectively antibody and busulfan injection). In addition, bone marrow examination of mice injected with monoclonal anti-PACAP or anti-VPAC1 antibodies revealed an increase in megakaryocyte numbers and showed a marked expansion and mobilization of megakaryocyte progenitor cells. Mice injected with a monoclonal anti-VPAC1 antibody showed an increase of about 50% in bone marrow CFU-MK. In conclusion, we provide evidence that the VPAC1 pathway modulates normal megakaryopoiesis. Further studies are needed to evaluate whether this pathway can be safely manipulated in man in the treatment of thrombocytopenia.

Prolonged Administration of Granulocyte Colony-Stimulating Factor (Filgrastim) to Patients With Fanconi Anemia: A Pilot Study

This report examines the effect of filgrastim (granulocyte colony-stimulating factor, [G-CSF] in 12 patients with neutropenia [absolute neutrophil count [ANC] < 1,000/mm3]) caused by Fanconi anemia (FA). Two of 14 patients who were evaluated for study entry were ineligible because of unsuspected cytogenetic abnormalities in their bone marrow (BM). G-CSF was started at 5 micrograms/kg/d. All patients had an increase in their ANC at week 8 (mean increase = 15,664/mm3). The median ANC during therapy was 5,030/mm3. Eight of 10 patients who completed 40 weeks on study maintained an ANC > 1,500/mm3 on G-CSF given every-otherday. Four patients had an increase in their platelet count by week 8 without transfusion (maximum increase = 23,000 to 45,000/mm3); however, platelet counts fell toward baseline levels as the G-CSF dose was reduced. BM CFU-MK were increased at week 8 in three of four evaluable patients. Four patients who did not receive red blood cell transfusions had an increase in their hemoglobin level of at least 2.0 g/dL. A fifth patient had a red blood cell transfusion in week 2 and then had a similar increase in hemoglobin level without subsequent transfusion. Eight of 10 patients who completed 40 weeks of treatment showed increases in the percentage of BM CD34+ cells measured by flow cytometry. The same proportion showed increases in peripheral blood CD34+ cells. Increased BM cellularity and myeloid hyperplasia were constant findings and were associated with increased expression of the proliferating cell nuclear antigen. Adverse experiences were mild fever (1 patient) and a new BM cytogenetic abnormality at week 40 (1 patient). This study shows that prolonged administration of G-CSF exerts a stimulatory effect on the BM of FA patients and may be used to maintain a clinically adequate ANC in these patients. G-CSF has beneficial effects on multiple hematopoietic lineages in some patients and may be a good candidate for use in combination cytokine protocols for FA patients with progressive aplastic anemia. G-CSF use results in an increase in circulating CD34+ cells, a finding with important implications for future gene transfer protocols.

Prolonged administration of granulocyte colony-stimulating factor (filgrastim) to patients with Fanconi anemia: a pilot study

This report examines the effect of filgrastim (granulocyte colony- stimulating factor, [G-CSF] in 12 patients with neutropenia [absolute neutrophil count [ANC] &lt; 1,000/mm3]) caused by Fanconi anemia (FA). Two of 14 patients who were evaluated for study entry were ineligible because of unsuspected cytogenetic abnormalities in their bone marrow (BM). G-CSF was started at 5 micrograms/kg/d. All patients had an increase in their ANC at week 8 (mean increase = 15,664/mm3). The median ANC during therapy was 5,030/mm3. Eight of 10 patients who completed 40 weeks on study maintained an ANC &gt; 1,500/mm3 on G-CSF given every-otherday. Four patients had an increase in their platelet count by week 8 without transfusion (maximum increase = 23,000 to 45,000/mm3); however, platelet counts fell toward baseline levels as the G-CSF dose was reduced. BM CFU-MK were increased at week 8 in three of four evaluable patients. Four patients who did not receive red blood cell transfusions had an increase in their hemoglobin level of at least 2.0 g/dL. A fifth patient had a red blood cell transfusion in week 2 and then had a similar increase in hemoglobin level without subsequent transfusion. Eight of 10 patients who completed 40 weeks of treatment showed increases in the percentage of BM CD34+ cells measured by flow cytometry. The same proportion showed increases in peripheral blood CD34+ cells. Increased BM cellularity and myeloid hyperplasia were constant findings and were associated with increased expression of the proliferating cell nuclear antigen. Adverse experiences were mild fever (1 patient) and a new BM cytogenetic abnormality at week 40 (1 patient). This study shows that prolonged administration of G-CSF exerts a stimulatory effect on the BM of FA patients and may be used to maintain a clinically adequate ANC in these patients. G-CSF has beneficial effects on multiple hematopoietic lineages in some patients and may be a good candidate for use in combination cytokine protocols for FA patients with progressive aplastic anemia. G-CSF use results in an increase in circulating CD34+ cells, a finding with important implications for future gene transfer protocols.

Megakaryocyte progenitors in paroxysmal nocturnal haemoglobinuria are sensitive to complement.

We have investigated growth in vitro of bone marrow megakaryocytic progenitors (CFU-Mk) in 7 patients with paroxysmal nocturnal haemoglobinuria (PNH) to determine the sensitivity of CFU-Mk to complement. Bone marrow light density mononuclear cells were exposed to fresh or heat-inactivated AB human serum in the presence of medium or isotonic sucrose solution. We found that the proliferative activity of bone marrow CFU-Mk in PNH patients was significantly lower than in controls. In addition, the number of CFU-Mk in PNH bone marrow cells exposed to isotonic sucrose and complement was reduced to 25% of that in PNH cells exposed to isotonic sucrose without complement. In conclusion, our finding showed an increased sensitivity of CFU-Mk in PNH bone marrow cells to complement, supporting the hypothesis that the PNH defect is present at the level of CFU-Mk.

Regulation of megakaryocyte colony forming cell numbers and ploidy by dideoxynucleosides in immunodeficient mice

We have recently demonstrated that azidothymidine (AZT) elevates the levels of circulating platelets in mice made immune deficient by infection with LP-BM5 murine leukemia virus (MAIDS mice). In an attempt to elucidate the mechanisms of the AZT platelet elevating effect, we examined the number of splenic and bone marrow megakaryocyte colony-forming cells (CFU-mk) and the ploidy of megakaryocytes derived from CFU-mk using fluorescence cytophotometric methods. Two other dideoxynucleosides (ddN) 2'3'-dideoxyinosine (ddl) and 2'3'-dideoxycytidine (ddC) were assessed to determine the specificity of the effect of AZT. MAIDS mice were given ddN in drinking water for 15 days. AZT was the only ddN that significantly increased circulating platelet levels in MAIDS mice. AZT significantly increased splenic CFU-mk in MAIDS mice, but bone marrow CFU-mk were not affected. ddl and ddC failed to change either platelet levels or the numbers of splenic or bone marrow CFU-mk. The ploidy of megakaryocytes derived from splenic and bone marrow CFU-mk were examined by first identifying CFU-mk by staining for acetylcholinesterase, followed by nuclear staining with propidium iodide. The fluorescence of individual cells was then measured using a Perceptics image analysis system. Modal ploidy of CFU-mk megakaryocytes derived from spleen cells of AZT-treated immunodeficient mice was shifted upwards from 16N to 32N. Similarly, AZT treatment changed the modal ploidy number of colony megakaryocytes derived from bone marrows of immunodeficient mice from 16N to 32N. The ploidy distribution was also significantly shifted. ddl and ddC failed to significantly alter either modal ploidy number or distribution of megakaryocytes derived from splenic or bone marrow CFU-mk. These findings suggest that AZT may affect physiological processes that lead to platelet formation.

In vivo and in vitro inhibitory effect of alpha-interferon on megakaryocyte colony growth in essential thrombocythaemia.

Megakaryocyte (MK) colony growth of bone marrow mononuclear non-adherent cells was evaluated in 28 patients with essential thrombocythaemia (ET) and in 26 normal controls. The number of MK-colony forming units (CFU-MK per 3 x 10(5) plated cells) was similar in ET (68 +/- 33) and in controls (63 +/- 37), independently of bone marrow accessory cells. On the contrary, the size of the MK colonies was significantly (P less than 0.01) greater in ET patients. Human recombinant alpha-interferon 2a (alpha-IFN), administered to 10 patients at a dose of 3 x 10(6) IU/d s.c. for 11 +/- 3 weeks, was capable of inducing a significant (P less than 0.01) decrease in the number (from 72 +/- 16 to 31 +/- 14) and size of bone marrow CFU-MK, together with a significant reduction of the platelet count (from 1031 +/- 325 to 378 +/- 75 x 10(9)/l). When added in vitro at time 0 to the culture dishes, alpha-IFN inhibited the CFU-MK growth of both normal and ET bone marrow samples, even at very low concentrations (1 and 10 IU/ml). This study demonstrates that alpha-IFN, both in vivo and in vitro, exerts an inhibitory effect on the growth of MK progenitors, which appears to correlate with the clinically documented antiproliferative effect of this cytokine.

Megakaryocyte progenitors in the bone marrow and peripheral blood of patients with myeloproliferative diseases.

We studied the behavior in culture of megakaryocyte progenitor cells (CFU-Mk) from peripheral blood (PB) and bone marrow (BM) cells in eight patients with myeloproliferative diseases (MPD). In seven patients we observed megakaryocyte (Mk) colony formation from PB cells, which were generated in the absence of any added stimulator and which did not increase after the addition of a source of Mk-colony stimulating activity (CSA-Mk). The number of BM CFU-Mk was significantly higher in patients than in controls, and in seven out of eight patients the responsiveness to added CSA-Mk was retained. Plasma obtained from six patients did not stimulate normal donors' BM target cells to form Mk colonies. These data demonstrate an expansion of the CFU-Mk pool in MPD patients without increased plasma levels of CSA-Mk, and suggest that PB and BM CFU-Mk of MPD patients might have different kinetic properties.