| BackgroundEssential thrombocythemia (ET) is a chronic myeloproliferative neoplasm (MPN) characterized by stem cell-derived clonal myeloproliferation with constantly high platelet count and an increased risk of vascular complications. Scientists have found that Janus kinase 2 (JAK2), calreticulin (CALR), or myeloproliferative leukemia virus oncogene (MPL) mutations occur in approximately 55%,25%, and 3% of ET patients, respectively. However,20% of patients with ET might be negative for all three mutations.This indicates that the molecular pathogenesis of ET is not fully understood.Platelet-derived growth factor (PDGF) was initially purified from platelet and characterized as a mitogen for fibroblasts and smooth muscle cells. PDGF and their receptors are associated with proliferative disorders, such as solid tumors and myeloproliferative diseases. In the hematopoietic system, genetic studies have reported that knock-out mice for PDGF-B or PDGFR-P showed anemia, thrombocytopenia. PDGF also stimulates the proliferation of megakaryocytes, erythrocytes, leukocytes, and their progenitors, and enhance the expansion of megakaryocyte progenitors from human CD34+ cells. PDGF is likely to be an autocrine factor that megakaryocytes and platelets not only make PDGF and express PDGF receptors, but also can be regulated by PDGF. More importantly, scientists had found PDGF-BB enhances the platelet recovery in irradiationinduced thrombocythemia in mice. As mentioned above, PDGF and their receptors have a megakaryocytopoiesis effect in human, but role of PDGF and their receptors in platelet disorders is much less reported.Imatinib, as a tyrosine kinase inhibitor, are firstly to be used in the treatment of BCR-ABL positive leukemia. In recent years, with the understanding of the role of tyrosine kinase inhibitors, scients found it is also effective to vrious solid cancers and MPNs with PDGFR mutations. PDGFR is a member of the tyrosine kinase family, and the biological effects of PDGF can be blocked by imatinib. Imatinib is widely used for the treatment of MPN with fusion of the FIP1L1 and PDGFRA, PDGFRB or FGFR1 gene. However its effect in essential thrombocytopenia has not been reported.Design and Methods1.PatientsAll patients met the clinical criteria of essential thrombosis with increased platelet numbers to 450 ×109in the peripheral blood and excluding other secondary thrombocythemia. Patients were first visited to department of hematology and not on treatment at the time of sample acquisition. Bone marrow blood specimens from ET patients were obtained and informed consents signed for sample collection. Normal controls were bone marrow donors from the southern hospital. Bone marrow platelet-poor plasma was obtained by separation, as described reviously. Bone marrow cells were obtained by hematolysis, washing and separation.2.Enzyme-linked immunosorbent assay PDGF-BB protein levels in bone marrow platelet-poor plasma samples from ET patients and normal controls were determined using Human PDGF-BB Quantikine enzyme-linked immunosorbent assay (ELISA) Kit (Sigmal-alodrich, Catalog Number RAB0397) following the manufacturer’s protocol. Samples were diluted five times according to the concentration of PDGF-BB in human blood plasma according to previous reporets. Samples were assayed in triplicate.3.Nucleated cell enumeration with PDGFR-β using flow cytometryAfter getting nucleated cells, the nucleated cells were washed twice in PBS and re-suspended at 1 × 107cells/ml in PBS. CD140b-PE (BD, Biosciences, San Jose, USA,558821) was added at the manufacturers’recommended concentrations and the cells were incubated with the antibodies for 30 minutes in the dark at room temperature. After staining, the cells were fixed with 0.5% paraformaldehyde and stored at 4℃ until fluorescence-activated cells sorting (FACS) were performed.4.The effects of PDGF on the platelet in miceFeale Balb/c mice (7 or 8 weeks old) were obtained from Medical Laboratory Animal Center of Guangdong (guangzhou, China). Ethical permission for the studies was granted. Animals were divided into four groups:a control group, a PDGF treatment group, a PDGF combining imatinib treatment group, an imatinib group. Mice were injected intraperitoneally with PDGF-BB (1.5ug/kg/day) (PeproTech, NJ, USA), or Imatinib (50mg/kg/day) (Novartis, MO, USA) or saline. Peripheral blood platelets were counted in blood samples collected on days 0,7, and 21. Mice were given free access to food and water and sacrificed on day 21.5. Bone marrow histology Bone marrow samples from four groups of mice were collected on day 21, after the mice had been sacrificed. The samples were fixed using 4% poly formaldehyde and cut into 5-μm sections. The slides were stained with Wright-Giemsa. Sections were observed under twenty-five high-power (400 X) and photographs were taken for analysis. The number of megakaryocytes and their morphological changes were examined.6.Activating JAK2/STAT3 pathway analysis of CHRF-288 cells by flow cytometryCHRF-288 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum as described previously. Cells were starved and pre-incubated with imatinib (5 mM) (Novartis, MO, USA) for 60 min, and then stimulated with PDGF-BB (200 ng/mL) for 30-60 mins. Cells from different treatment groups were collected and stained with anti-phospho-JAK2 (P-JAK2) antibody (CST, cell signaling,8082) or anti-phospho-STAT3/(P-STAT3) (BD, Biosciences, San Jose, USA,557814) after fixation and permeabilization using the Cytofix Fixation Buffer (BD, Biosciences, San Jose, USA,554655) and Phosflow Perm Buffer III (BD, Biosciences, San Jose, USA,558050), according to the manufacturer’s instructions. The cells stained with anti-phospho-JAK2 (P-JAK2) antibody would stain with the second antibody (Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor(?) 488 conjugate, thermo scientific, A-11008) subsequently. Ten thousand events were acquired for each sample and the population of P-JAK2 and P-STAT3 positive cells was analyzed using flow cytometry.6.Activating PI3K/AKT pathway analysis of CHRF-288 cells by western blotCHRF-288 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum as described previously. Cells were starved and pre-incubated with imatinib (5 mM) (Novartis, MO, USA) for 60 min, and then stimulated with PDGF-BB (200 ng/mL) for 30-60 mins. Cells from different treatment groups were collected and cells were washed with ice-cold phosphate-buffered saline and lysed. Protein oncentrations were determined by the Bradford protein assay. Proteins were then loaded on sodium dodecyl sulfate polyacrylamide gels and separated by electrophoresis. The gels were transferred to nitrocellulose membrane and blocked with 5% bovine serum albumin in Tris-buffered saline solution containing 0.1% Tween-20. Primary antibodies, phospho-AKT (Cell Signaling, MA, USA) were incubated overnight at 4℃. After washing, the membrane was incubated with secondary antibody, goat antirabbit antibody (Santa Cruz, CA, USA). Total Akt and phospho-Akt were visualized by detection systems.Results1. The expression of PDGF-BB and PDGFR-β in ET patients and normal controlsTo determine circulating levels of PDGF-BB, platelet-poor plasma samples from ET patients (n=16) and normal controls (n=8) were examined in this study. We found an increased PDGF-BB level in ET patients (2070.92±123.98 pg/ml), compared to normal controls (1381.85±128.37pg/ml) (P=0.002).We tested the expression of PDGFR-β in bone marrow cells from ET patients (n=6) and normal controls (n=3) by flow cytometry, and found that the expression of PDGFR-β were significantly higher in cells from the bone marrow of ET patients than the levels expressed in that of normal controls.2. In vivo effects of PDGF on peripheral blood cell counts in miceOn day 0, the basal numbers of peripheral blood platelets were 452 ×109/L, 470×109/L,454×109/L and 436×109/L in the control group, PDGF group, Imatinib group and PDGF combining Imatinib group, and there was no difference in platelet counts among the four groups. At day 21, the PDGF group had significantly higher platelet counts than control groups (537±15×109/L versus454±9×109/L, n=4, P=0.006). In a further investigation for the impact of imatinib to the thrombocytopoiesis of PDGF, we assessed the platelet counts in the PDGF alone group and PDGF combining imatinib group. We found that the platelet counts was significantly lower in the PDGF combining imatinib group than the PDGF alone group (424±25×109/L versus 537±15×109/L, n=4, P=0.001) at day 21, however the platelet counts in imatinib group were similar to the controls at day 21, despite it was lower than control group at day 7. The data suggest the effect of promoting platelet of PDGF was abrogated by adding imatinib. Above results showed that PDGF have a thrombocytopoiesis effect in vivo, and the effect can be blocked by PDGFR-β inhibitor.3. Effects of PDGF on mice bone marrow histologyThe bone marrow histology from differently treated mice were collected on day 21 and stained with Wright-Giemsa for histological examination. Slips were analyzed under a microscope at high-power (400 X). Compared to the control group, there were more megakaryocytes in the PDGF-treated mice, revealing PDGF-evoked ET by promoting megakaryocytopoiesis. However, in the PDGF combined imatinib treated mice, many apoptotic megakaryocytes were observed, and the number of megakaryocytes was significantly lower than that in PDGF-treated mice, indicating imatinib can block the effect of PDGF on megakaryocytes. These data suggest that PDGF significantly enhanced the hematopoiesis of the megakaryocytic lineage, and the effect can be inhibited by imatinib.4. Thrombopoiesis of PDGF may be mediated via PDGFR and JAK2/STA3, PI3K/AKT signal pathway subsequentlyTo determine whether JAK2/STAT3 is involved in PDGF-induced megakaryocytopoiesis, nutrition-depleted CHRF-288 cells were incubated with PDGF-BB (200 ng/mL). Cells were then stained with anti-P-JAK2 or anti-P-STAT3 antibody. PDGF-BB increased the expression of P-JAK2, P-STAT3 and P-STAT5, indicating JAK2/STAT3 and PI3K/AKT activation. In a further investigation for an upstream executor that triggers the JAK2/STAT3 and PI3K/AKT pathway, CHRF-288 cells were treated with PDGF alone or PDGF plus imatinib. PDGF alone increased the production of P-JAK2, P-STAT3 and P-AKT, while the addition of imatinib compromised the effects of PDGF. However, imatinib alone did not affect the expression of P-JAK2, P-STAT3 and P-AKT. This indicates that PDGF triggers the JAK2/STAT3 and PI3K/AKT pathway via PDGFR-β, and that imatinib can block this effect.ConclusionIn this study, we report that the expressions of PDGF-BB and PDGFR in essential thrombocythemia patients are higher than normal controls, and PDGF effectively promote thrombocytopoiesis in PDGF overe-expression mice. We further demonstrated that this effect is likely to be mediated via PDGF receptors with subsequent activation of the JAK2/STAT3 pathway, which inhibits apoptosis in megakaryocytes. And imatinib can abolish the effect in vivo via blocking PDGFR. |
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