Cell Line MEG-01 Research Articles

Anti-Diabetes, Anti-Gout, and Anti-Leukemia Properties of Essential Oils from Natural Spices Clausena indica, Zanthoxylum rhetsa, and Michelia tonkinensis.

Essential oils (EOs) of Clausena indica fruits, Zanthoxylum rhetsa fruits, and Michelia tonkinensis seeds were analyzed for their phytochemical profiles and biological activities, including anti-diabetes, anti-gout, and anti-leukemia properties. Sixty-six volatile compounds were identified by gas chromatography–mass spectrometry (GC–MS), in which, myristicin (68.3%), limonene (44.2%), and linalool (49.3%) were the most prominent components of EOs extracted from C. indica, Z. rhetsa, and M. tonkinensis, respectively. In addition, only EOs from C. indica inhibited the activities of all tested enzymes comprising α-amylase (IC50 = 7.73 mg/mL), α-glucosidase (IC50 = 0.84 mg/mL), and xanthine oxidase (IC50 = 0.88 mg/mL), which are related to type 2 diabetes and gout. Remarkably, all EOs from C. indica, Z. rhetsa (IC50 = 0.73 mg/mL), and M. tonkinensis (IC50 = 1.46 mg/mL) showed a stronger anti-α-glucosidase ability than acarbose (IC50 = 2.69 mg/mL), a known anti-diabetic agent. Moreover, the growth of leukemia cell Meg-01 was significantly suppressed by all EOs, of which, the IC50 values were recorded as 0.32, 0.64, and 0.31 mg/mL for EOs from C. indica, Z. rhetsa, and M. tonkinensis, respectively. As it stands, this is the first report about the inhibitory effects of EOs from C. indica and Z. rhetsa fruits, and M. tonkinensis seeds on the human leukemia cell line Meg-01 and key enzymes linked to diabetes and gout. In conclusion, the present study suggests that EOs from these natural spices may be promising candidates for pharmaceutical industries to develop nature-based drugs to treat diabetes mellitus or gout, as well as malignant hematological diseases such as leukemia.

Tanshinone Iia Inhibits Megakaryopoiesis in Immune Vasculitis

Introduction Tanshinone IIA, an active component of Danshen (Salvia miltiorrhiza), has been used for centuries to treat hypercoagulation-related diseases, which attributed to its anti-platelet and anti-inflammatory effects. However, the role of Tanshinone IIA in megakaryocytes, the precursor of platelet within the bone marrow, remains unclear. Therefore, the present study established a rabbit model with immune vasculitis to examine the effect of Tanshinone IIA on megakaryopoiesis and to identify the underlying mechanism(s).Methods Immune vasculitis was established in rabbits (3-4 weeks old) by two intravenous injection of 10% bovine serum albumin (2.5 ml/kg) at two-week interval. Those rabbits were randomly treated with Tanshinone IIA (5 mg/kg/d, 7 d, iv) or aspirin (100 mg/kg/d, 7 d, ig). Megakaryocyte count and CFU-MK formation were measured by Wright's and AChE staining, respectively. Human megakaryotic cell lines Meg-01 and CHRF-288-11 were used to examine the effect of Tanshinone IIA on apoptosis by Annexin V-FITC/PI, mitochondrial membrane potential/JC-1 and Caspase-3 activity assays using flow cytometry.ResultsIn rabbits with immune vasculitis, the platelet count, platelet aggregation and the serum levels of inflammatory cytokines IL-1β, TNF-α and IL-6 were significantly increased when compared to their healthy controls. After 7 days of Tanshinone IIA treatment, all these parameters were significantly reduced, with the inhibitions comparable to those caused by aspirin. In addition, the number of megakaryocytes and the formation of CFU-MK were also statistically increased in rabbits with immune vasculitis, which could be significantly reduced by Tanshinone IIA. In vitro, Tanshinone IIA (1, 3, 10 and 30 μg/ml) also significantly inhibited the formation of CFU-MK of bone marrow cells of BALB/c mice (6-10 weeks) in a dose-dependent manner. In human megakaryocytic cell line Meg-01, Tanshinone ⅡA (10 μg/ml, 72 h) induced apoptosis; both early and late apoptotic rates were significantly increased. In another human megakaryocytic cell line CHRF-288-11, Tanshinone ⅡA (10 μg/ml, 72 h) statistically increased the proportion of depolarized cells, from 9.70% to 14.13%, according to mitochondrial membrane potential using JC-1 assay. The expression of active Caspase-3 in CHRF-288-11 was also significantly increased by Tanshinone ⅡA (10 μg/ml, 72 h) from 5.25% to 15.86%.Conclusion The present study shows that Tanshinone IIA ameliorates immune vasculitis by inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes, which might explain the anti-platelet and anti-inflammatory effects of Tanshinone IIA. DisclosuresNo relevant conflicts of interest to declare.

Effect of Thrombospondin-1 on Apoptosis of Human Megakaryocytic Leukemia Cells

Background: Thrombospondin 1 (TSP-1) is an extracellular matrix protein that interacts with a wide array of ligands including cell receptors, growth factors, cytokines, and proteases to regulate various physiological and pathological processes. TSP-1 induces apoptosis of endothelial and cancer cells via its receptor CD36. This study was to investigate the effect of TSP-1 on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism.Methods: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations, to investigate the effect of TSP-1 on the growth of megakaryocytes. The early apoptosis and the activity of the apoptotic protease caspase-3 were detected by flow cytometry. Bone marrow cells of mice were cultured for CFU-MK and stained with acetylcholine to detect the differentiation of megakaryocytes.Results: CD36 antigen was detected on the surface of Meg-01 cells by flow cytometry and immunocytochemistry. TSP-1 (5 μg/mL) inhibited the proliferation of Meg-01 cells, but not M-07e cells (CD36 -). After the addition of CD36 antibody FA6-152 (5, 10 and 25 μg/mL), the inhibitory effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5 and 7.5 μg/mL) exerted a pro-apoptotic effect by increasing the expression of Annexin V (P<0.01) and caspase-3 activation (P<0.01). Addition of FA6-152 (25 μg/mL) can significantly reduce the apoptosis induced by TSP-1 in Meg-01 cells. In addition, TSP-1 (5, 10 and 25 μg/mL) repress the formation of CFU-MK in mouse bone marrow cells, while β-TG did not. This repression was relieved efficiently by FA6-152 (25 μg/mL).Conclusion: TSP-1 could inhibit the proliferation of Megakaryocyte cell line Meg-01 cells, and induce it's apoptosis. TSP-1 may induce apoptosis of Meg-01 cells via CD36 or caspase-3, which provides a potential new drug choice for clinical treatment of megakaryocytic leukemia. DisclosuresNo relevant conflicts of interest to declare.

JMJD1C knockdown affects myeloid cell lines proliferation, viability, and gemcitabine/carboplatin-sensitivity.

Chemotherapy-induced hematological toxicities are potentially life-threatening adverse drug reactions that vary between individuals. Recently, JMJD1C has been associated with gemcitabine/carboplatin-induced thrombocytopenia in non-small-cell lung cancer patients, making it a candidate marker for predicting the risk of toxicity. This study investigates if JMJD1C knockdown affects gemcitabine/carboplatin-sensitivity in cell lines. Lentiviral transduction-mediated shRNA knockdown of JMJD1C in the cell lines K562 and MEG-01 were performed using shRNA#32 and shRNA#33. The knockdown was evaluated using qPCR. Cell proliferation, viability, and gemcitabine/carboplatin-sensitivity were subsequently determined using cell counts, trypan blue, and the MTT assay. ShRNA#33 resulted in JMJD1C downregulation by 56.24% in K562 and 68.10% in MEG-01. Despite incomplete knockdown, proliferation (reduction of cell numbers by 61-68%, day 7 post-transduction) and viability (reduction by 21-53%, day 7 post-transduction) were impaired in K562 and MEG-01 cells. Moreover, JMJD1C knockdown reduced the gemcitabine IC50-value for K562 cells (P < 0.01) and MEG-01 cells (P < 0.05) compared to scrambled shRNA control transduced cells. Our results suggest that JMJD1C is essential for proliferation, survival, and viability of K562 and MEG-01 cells. Further, JMJD1C also potentially affects the cells gemcitabine/carboplatin-sensitivity. Although further research is required, the findings show that JMJD1C could have an influential role for gemcitabine/carboplatin-sensitivity.

Inhibition of Cell Cycle Progression, Induction of Apoptosis, and Changes in Surface Markers of MEG-01 Megakaryoblastic Cells Exposed to a Random Positioning Machine

One cause of thrombocytopenia in astronauts after spaceflight is decreased platelet production. To increase our understanding of thrombopoiesis in humans while in space, we investigated the effects of simulated microgravity, achieved using a random positioning machine (RPM), on megakaryoblastic cells (MEG-01 cell line). Exposure of MEG-01 cells to simulated microgravity for up to one week significantly increased cellular apoptosis compared to the static group (1 g-control). Flow cytometry analysis of the cell cycle revealed a significant increase in the percentage of cells in the G0/G1 phase after one week of RPM-exposure compared to the static group. Additionally, after one week, a difference in morphology was detected between the cells of the static group and the cells exposed to microgravity conditions. The expression of the CD33 surface marker was significantly decreased after a one week of microgravity exposure compared to the 1 g-control. We, therefore, concluded that in MEG-01 cells, simulated microgravity induces apoptosis, inhibits cell cycle progression of cells from G0/G1 into S phase, decreases cell proliferation, and decreases the expression of surface markers. We believe that, with insufficient physiological compensation, these changes under microgravity conditions may lead to disorders of megakaryocytes differentiation and/or decreased platelet production. It should also be noted that the human cell line MEG-01 could be a useful model for studying the effects of simulated microgravity on platelet production because of their ability to generate platelet-like particles.

An in Vitro Study on the Role of Angiogenesis in Iron Deficiency Induced Reactive Thrombocytosis

Introduction: Iron deficiency (ID) is one of the recognized causes of reactive thrombocytosis in children. Factors that are commonly associated with megakaryopoiesis such as thrombopoietin (TPO), interleukin 6 (IL-6) and IL-11 are not altered in patients with iron deficiency and thrombocytosis suggesting the role of alternate mechanisms in controlling this process. We have previously shown using an ID rat model that ID increased the number of megakaryocytes in the bone marrow. We have also shown an increase in VEGFR (FLT1) and CXCR4 staining in bone marrow slides of ID rats. This data suggests that angiogenesis plays a vital role in the development of reactive thrombocytosis in response to ID. In this report, we have expanded our study to identify specific angiogenic signaling molecules associated with ID and used functional assays to validate it.Methods: For this study, we used the megakaryoblast cell line MEG-01 as an in vitro model of megakaryopoiesis. MEG-01 cells were adapted to grow in chemically defined serum free medium containing iron (iron replete media). For iron deficiency, serum free iron free media was mixed with iron replete media at a 1% v/v concentration (iron deplete media). For our experiments, MEG-01 cells were grown in both iron replete and depleted media for 7 days. Cell viability was measured using the trypan blue exclusion assay. Messenger-RNA expression of iron-related markers (TFR1, TFR2, FLT1, FLT3, FTL, FTH1, TF, HMOX1 and HMOX2) and angiogenic markers (VEGFA, VEGFB, VEGFC, PDGF, ANGPTL1, ANGPTL2, FGF2) was studied using real time PCR. We performed functional validation of angiogenesis with an in vitro tube formation assay using human umbilical vein endothelial (HUVEC) cells. For statistical analysis of the data we performed the t test using graph pad prism software and we considered p<0.05 as statistically significant.Results: In low iron conditions, MEG-01 cells showed a significant increase in FLT1 (4 fold) and FLT3 (3 fold) expression using real time PCR (p<0.001). Iron deficiency also induced a 2 fold increase in the mRNA expression of angiogenic molecules VEGFB, VEGFC, FGF2 and PDGFA (p<0.001). Using the tube formation assay, we also show that conditioned media collected from iron deficient MEG-01 cells induced increased vessel formation in endothelial cells.Conclusion: In this study, we were able to validate our earlier in vivo findings on iron deficiency induced reactive thrombocytosis. We show that cells adapt to low iron conditions by upregulating FLT1, FLT3 and FTL. We also show that several markers in the angiogenesis pathway like VEGFB, VEGFC, FGF2 and PDGFA are upregulated in response to iron deficiency. We were also able to show that an increase in these angiogenic molecules induced increased vessel formation in endothelial cells. This report, along with our previous findings, points to the importance of the angiogenic pathway in reactive thrombocytosis induced by iron deficiency. DisclosuresNo relevant conflicts of interest to declare.

Cyanidin-3-o-β-Glucoside Induces Megakaryocyte Apoptosis via PI3K/Akt- and MAPKs-Mediated Inhibition of NF-κB Signalling.

Apoptotic-like phase is an essential step in thrombopoiesis from megakaryocytes. Anthocyanins are natural flavonoid pigments that possess a wide range of biological activities, including protection against cardiovascular diseases and induction of tumour cell apoptosis. We investigated the effects and underlying mechanisms of cyanidin-3-o-β-glucoside (Cy-3-g, the major bioactive compound in anthocyanins) on the apoptosis of human primary megakaryocytes and Meg-01 cell line in vitro. We found that Cy-3-g dose-dependently increased the dissipation of the mitochondrial membrane potential, caspase-9 and caspase-3 activity in megakaryocytes from patients with newly diagnosed acute myeloid leukaemia but not in those from healthy volunteers. In Meg-01 cells, Cy-3-g regulated the distribution of Bak, Bax and Bcl-xL proteins in the mitochondria and cytosol, subsequently increasing cytochrome c release and stimulating caspase-9 and caspase-3 activation and phosphatidylserine exposure. However, Cy-3-g did not exert significant effects on factor-associated suicide (Fas), Fas ligand, caspase-8 or Bid expression. Cy-3-g inhibited nuclear factor kappa B (NF-κB) p65 activation by down-regulating inhibitor of NF-κB kinase (IKK)α and IKKβ expression, followed by the inhibition of inhibitor of NF-κB (IκB)α phosphorylation and degradation and subsequent inhibition of the translocation of the p65 sub-unit into the nucleus, and finally stimulating caspase-3 activation and phosphatidylserine exposure. The inhibitory effect of Cy-3-g on NF-κB activation was mediated by the activation of extracellular signal-regulated kinases (Erk1/2) and p38 mitogen-activated protein kinase (MAPK) and the inhibition of phosphoinositide 3-kinase (PI3K)/Akt signalling. U0126 (Erk1/2 inhibitor), SB203580 (p38 MAPK inhibitor) and 740 Y-P (PI3K agonist) significantly reversed Cy-3-g-reduced phosphorylation of p65. Taken together, our data indicate that Cy-3-g induces megakaryocyte apoptosis via the inhibition of NF-κB signalling, which may play important roles in regulating thrombopoiesis.

Identification of eNOS‐based Megakaryocyte Subpopulations and Their Pharmacological Characterization by IFNγ and IL‐10

PurposeRecently, our laboratory identified in human blood two platelet subpopulations based on the presence or absence of a functional endothelial nitric oxide synthase (eNOS)‐signalling pathway and the ability to produce nitric oxide (NO). We have found that eNOS‐negative (eNOSneg) platelets, although less abundant are more reactive than eNOS‐positive (eNOSpos) platelets and initiate aggregate/thrombus formation, while eNOS‐positive platelets through their ability to generate NO limit aggregate size. As platelets derive from bone marrow megakaryocytes, we hypothesized that eNOSneg and eNOSpos subpopulations of megakaryocytes exist and give rise to their respective eNOS‐based platelet subpopulations. Additionally, we hypothesized that interferon‐γ (IFNγ) and interleukin‐10 (IL‐10), which are known to counter‐regulate eNOS expression and megakaryocyte differentiation, would determine in a concentration manner the ratio of eNOSneg to eNOSpos megakaryocytes.MethodsThe human megakaryoblastic cell line (Meg‐01) and megakaryocytes isolated from eNOS‐GFP transgenic mice were studied. RT‐PCR and DNA sequencing were performed to validate eNOS presence within Meg‐01. Flow cytometry was used to identify eNOSneg and eNOSpos megakaryocytes, and NO production was measured using DAF‐FM, a cell‐permeable fluorescent probe. Further, Meg‐01 cells were treated for 48 hours with IFNγ (0 – 100 ng/ml) and IL‐10 (0 – 100 ng/ml), and eNOS as well as inducible NOS (iNOS) were measured by flow cytometry and immunoblot, respectively.ResultsThe presence of eNOS was confirmed in Meg‐01 and eNOS‐GFP transgenic mice. Similar to human platelets, the majority of Meg‐01 cells were eNOSpos (91.5%±1.2 vs. 8.5%±1.2 eNOSneg, P&lt;0.05) and NO‐producing (92.0%±3.34 vs. 8.0%±6.68 non‐producing, P &lt; 0.05). Conversely, similar to mouse platelets, the majority of mouse megakaryocytes were eNOSneg (95.8%±0.7 vs. 4.2%±0.7 eNOSpos, P &lt; 0.05). The proinflammatory cytokine IFNγ decreased the percent eNOSpos Meg‐01 in a concentration‐dependent manner and also increased the level of iNOS within Meg‐01. IL‐10 in a concentration‐dependent manner (10–100ng/ml) reversed IFNγ‐induced iNOS expression, but not the decrease in percent eNOSpos Meg‐01. The changes in eNOS expression within Meg‐01 were further confirmed by qPCR within the total Meg‐01 population and demonstrated that IFNγ (10 ng/ml) caused a 6.4 fold decrease of eNOS mRNA but only 2.2 fold decrease in the presence IL‐10 (100ng/ml).ConclusionseNOS‐based megakaryocyte subpopulations exist within the Meg‐01 cell line and in eNOS‐GFP transgenic mice. Similar to previously shown for platelets, major species differences exist in eNOSpos to eNOSneg ratios between human and mouse megakaryocytes. IFNγ decreases eNOS expression and percent eNOSpos Meg‐01; while increasing iNOS expression. However, high concentrations of IL‐10 attenuated this effect. Our data show that pro‐inflammatory IFN‐γ may promote growth/differentiation of megakaryocytes with decreased levels/absence of eNOS, which may lead to formation of higher numbers of more reactive eNOSneg platelets potentially leading to higher risk of a pro‐thrombotic state. Further experiments are required to confirm whether eNOSpos and eNOSneg megakaryocytes give rise to their respective platelet subtypes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thrombin‐reduced miR‐27b attenuates platelet angiogenic activities in vitro via enhancing platelet synthesis of anti‐angiogenic thrombospondin‐1

Background Platelets can synthesize proteins upon activation. Platelets contain a number of microRNAs (miRNA) and a fully functional miRNA effector machinery. It is, however, unclear if platelet miRNAs can regulate protein synthesis of platelets, and whether the regulation may produce a physiological impact. Objectives To investigate if and how platelet miRNAs regulate de novo syntheses of angiogenic regulators and subsequently modulate platelet angiogenic activities. Methods and Results Microarray-based miRNA profiling showed that thrombin stimulation in vitro down- or up-regulated a number of platelet miRNAs, both in the total platelet miRNAs and in Ago2-associated miRNAs. Among those altered miRNAs, miR-27b was down-regulated in both the total and Ago2-immunoprecipitated miRNA profiles of platelets, which was confirmed by reverse transcription-quantitative PCR (RT-qPCR). Using western blotting assays, we showed that thrombin induced platelet de novo synthesis of thrombospondin-1, and that the level of thrombospondin-1 synthesis could reach a level of 3-5-fold higher than that before thrombin stimulation. With either the platelet precursor megakaryocyte cell line MEG-01 cells or mature platelets, we demonstrated that transfection of miR-27b mimic, but not the negative control of miRNA mimic, markedly reduced thrombospondin-1 protein levels. The latter subsequently enhanced platelet-dependent endothelial tube formation on matrigel. Conclusions Thrombin stimulation in vitro reduces platelet miR-27b levels that may markedly enhance thrombin-evoked platelet de novo synthesis of thrombospondin-1. Elevation of platelet miR-27b by transfection inhibits thrombospondin-1 synthesis, and subsequently enhances platelet pro-angiogenic activities. Hence, platelet activation-dependent reduction of miR-27b levels may represent a novel negative regulatory mechanism of platelet angiogenic activities.

Forced expression of Wnt antagonists sFRP1 and WIF1 sensitizes chronic myeloid leukemia cells to tyrosine kinase inhibitors.

Chronic myeloid leukemia is a clonal myeloproliferative disorder that arises from the neoplastic transformation of the hematopoietic stem cell, in which the Wnt/β-catenin signaling pathway has been demonstrated to play an important role in disease progression. However, the role of Wnt signaling antagonists in therapy resistance and disease progression has not been fully investigated. We aimed to study the effects of Wnt/β-catenin pathway antagonists-secreted frizzled-related protein 1 and Wnt inhibitory factor 1-on resistance toward tyrosine kinase inhibitors in chronic myeloid leukemia. Response to tyrosine kinase inhibitors was analyzed in secreted frizzled-related protein 1 and Wnt inhibitory factor 1 stably transfected K562 cells. Experiments were repeated using a tetracycline-inducible expression system, confirming previous results. In addition, response to tyrosine kinase inhibitor treatment was also analyzed using the secreted frizzled-related protein 1 expressing, BCR-ABL positive MEG01 cell line, in the presence and absence of a secreted frizzled-related protein 1 inhibitor. Our data suggests that total cellular β-catenin levels decrease in the presence of secreted frizzled-related protein 1 and Wnt inhibitory factor 1, and a significant increase in cell death after tyrosine kinase inhibitor treatment is observed. On the contrary, when secreted frizzled-related protein 1 is suppressed, total β-catenin levels increase in the cell and the cells become resistant to tyrosine kinase inhibitors. We suggest that Wnt antagonists carry the potential to be exploited in designing new agents and strategies for the advanced and resistant forms of chronic myeloid leukemia.

The MAP3K Apoptosis Signal-Regulating Kinase 1 (ASK1) Functions As a Calcium-Dependent Regulator of Platelet Activation Downstream of the G(α)q-PLC Signaling Axis

Upon vascular injury, platelets rapidly adhere to sub-endothelial matrix proteins and activate to form a hemostatic platelet plug. Dysregulation of the molecular mechanisms dictating platelet plug formation is responsible for numerous thrombotic disorders. While it is known that MAPK signaling proteins such as p38, JNK, and ERK play a central role in platelet activation, little is known about the upstream MAP3K proteins regulating their activation following receptor stimulation. Our laboratory has identified the presence and activation of Apoptosis Signal-regulating Kinase 1 (ASK1), a member of the MAP3K family in human and mouse platelets. Ablation of Ask1 from mice results in a strong anti-thrombotic phenotype, and Ask1-/- platelets display significant functional defects such as integrin αIIbβ3 activation, granule secretion, and thromboxane A2 (TxA2) generation. To elucidate the signaling pathway leading to ASK1 activation, washed human platelets (4 x 108 platelets/mL) were evaluated for ASK1 phosphorylation at residue T845 (activated ASK1) by western blot analysis. Blocking TxA2 generation with aspirin (1 mM) severely diminished ASK1 activation induced by PAR1 peptide SFLLRN (100 μM), convulxin (10 ng/mL), and ADP (20 μM). Interestingly, PAR4 peptide AYPGKF (100 μM)-induced ASK1 activation was unaffected by aspirin treatment, suggesting AYPGKF-induced ASK1 activation to occur independently of TxA2 generation. The TxA2 mimetic, U46619, also induced strong T845 ASK1 phosphorylation. This data suggests ASK1 activation in human platelets to occur downstream of the TxA2 receptor TP-α and thrombin receptor PAR4. In nucleated cells, ASK1 is primarily activated by reactive oxygen species (ROS) following oxidative stress. The mechanism of ASK1 activation in platelets has never been studied. Treatment of washed human platelets (4 x 108 platelets/mL) with the ROS donor hydrogen peroxide (1 μM) failed to induce ASK1 activation. Additionally, scavenging of intracellular ROS by the ROS-scavenging compound MnTMPyP (200 μM) had no effect on agonist-induced ASK1 activation suggesting that ASK1 is activated by a ROS-independent mechanism. Interestingly, inducing intracellular calcium rise with the SERCA inhibitor, thapsigargin (1 µM), resulted in strong activation of ASK1 in human platelets. Blocking calcium rise using the calcium chelator BAPTA (50 μM) blocked both thapsigargin and agonist-induced ASK1 activation. This apparent dependency on calcium for ASK1 activation was also observed in the megakaryoblastic cell line MEG-01. Inhibition of IP3-mediated calcium release using the PLC inhibitor U73122 (10 μM) was sufficient to block ASK1 activation induced by U46619 (5 μM) and AYPGKF (100 μM), providing further evidence of a calcium-dependent mechanism for ASK1 activation in human platelets. The identification of ASK1 in platelets and the observed defects in Ask1-/- murine platelets as well as the impaired thrombosis highlights the central role of ASK1 in mediating platelet activation which, until this point, has largely been overlooked. In addition, the observation that ASK1 activation in platelets is exclusively calcium-dependent makes platelets to be a unique model for studying calcium-dependent ASK1 signaling that is independent of ROS. Collectively, we strongly believe that ASK1 is a novel target for anti-thrombotic drug therapy. DisclosuresNo relevant conflicts of interest to declare.

Casiopeina III-Ea, a copper-containing small molecule, inhibits the in vitro growth of primitive hematopoietic cells from chronic myeloid leukemia

Several novel compounds have been developed for the treatment of different types of leukemia. In the present study, we have assessed the in vitro effects of Casiopeina III-Ea, a copper-containing small molecule, on cells from patients with Chronic Myeloid Leukemia (CML). We included primary CD34+ Lineage-negative (Lin−) cells selected from CML bone marrow, as well as the K562 and MEG01 cell lines. Bone marrow cells obtained from normal individuals – both total mononuclear cells as well as CD34+ Lin− cells- were used as controls. IC50 corresponded to 0.5μM for K562 cells, 0.63μM for MEG01 cells, 0.38μM for CML CD34+ lin− cells, and 1.0μM for normal CD34+ lin− cells. Proliferation and expansion were also inhibited to significantly higher extents in cultures of CML cells as compared to their normal counterparts. All these effects seemed to occur via a bcr-abl transcription-independent mechanism that involved a delay in cell division, an increase in cell death, generation of Reactive Oxygen Species and changes in cell cycle. Our results demonstrate that Casiopeina III-Ea possesses strong antileukemic activity in vitro, and warrant further preclinical (animal) studies to assess such effects in vivo.

Platelet WDR1 suppresses platelet activity and is associated with cardiovascular disease

AbstractPlatelet activity plays a major role in hemostasis with increased platelet activity likely contributing to the pathogenesis of atherothrombosis. We sought to identify associations between platelet activity variability and platelet-related genes in healthy controls. Transcriptional profiling of platelets revealed that WD-40 repeat domain 1 (WDR1), an enhancer of actin-depolymerizing factor activity, is downregulated in platelet messenger RNA (mRNA) from subjects with a hyperreactive platelet phenotype. We used the human megakaryoblastic cell line MEG-01 as an in vitro model for human megakaryocytes and platelets. Stimulation of MEG-01 with thrombin reduced levels of WDR1 transcripts and protein. WDR1 knockdown (KD) in MEG-01 cells increased adhesion and spreading in both the basal and activated states, increased F-actin content, and increased the basal intracellular calcium concentration. Platelet-like particles (PLPs) produced by WDR1 KD cells were fewer in number but larger than PLPs produced from unmodified MEG-01 cells, and had significantly increased adhesion in the basal state and upon thrombin activation. In contrast, WDR1 overexpression reversed the WDR1 KD phenotype of megakaryocytes and PLPs. To translate the clinical significance of these findings, WDR1 expression was measured in platelet RNA from subjects with established cardiovascular disease (n = 27) and age- and sex-matched controls (n = 10). The WDR1 mRNA and protein level was significantly lower in subjects with cardiovascular disease. These data suggest that WDR1 plays an important role in suppressing platelet activity, where it alters the actin cytoskeleton dynamics, and downregulation of WDR1 may contribute to the platelet-mediated pathogenesis of cardiovascular disease.

Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor.

19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists.

Normalization of qPCR in platelets – YWHAE a potential genericreference gene

The mRNA of human platelets has been extensively studied and it is generally appreciated that platelets contain mRNA transcripts derived from the megakaryocytes, and they have the ability to translate it into proteins. Additionally, platelets contain microRNA (miRNA) that has been shown to potentially regulate the translation of certain proteins. When quantifying gene expression by quantitative real-time polymerase chain reaction (qPCR), a valid normalization method is required and the use of reference genes is a common and robust approach. It is recommended to perform a proper validation of potential reference genes for each individual experimental setup. Previous studies have mainly been performed using commonly used reference genes for nucleated cells, and to our knowledge there are no global evaluations of the stability of transcripts in platelets. Finding a stable transcript would be valuable for inter-study comparisons, and the aim of this study was to identify one or more stable mRNA transcripts suitable as generic reference genes for mRNA gene expression studies in platelets. Platelets were incubated for 24 h and microarray of platelet mRNA revealed that the levels of YWHAE, B2M, ITM2B, H3F3A, PF4V1 remained similar between 0 and 24 h. Further validation of the stability of these genes together with GAPDH, RN18S1, and PPIA, genes frequently used as reference genes in platelet studies, was performed using qPCR after different in vitro conditions. In addition, inter-individual stability of the genes was analyzed in diabetic patients compared with healthy matched controls. Analysis of gene stability by the software RefFinder revealed that YWHAE, PF4V1, and B2M were the most stable genes in platelets from healthy donors. In addition, YWHAE was stable between subjects. Furthermore, the potential influence of miRNA on the selected genes was investigated by knockdown of Dicer1 in the megakaryocytic cell line MEG01. YWHAE, H3F3A, B2M, and GAPDH remained unchanged over time in MEG01 cells indicating that these genes are not regulated by miRNA and hence are more stably expressed. In conclusion, YWHAE is a stable transcript in platelets and we suggest the use of YWHAE as a generic reference gene in mRNA gene expression studies.

Platelets constitutively express IL-33 protein and modulate eosinophilic airway inflammation

Although platelets play a key role in allergic inflammation in addition to their well-established role in hemostasis, the precise mechanisms of how platelets modulate allergic inflammation are not fully understood. IL-33 is an essential regulator of innate immune responses and allergic inflammation. We sought to determine the expression of IL-33 protein by platelets and its functional significance in airway inflammation. IL-33 protein in human platelets, the human megakaryocyte cell line MEG-01, and bone marrow-derived mouse megakaryocytes was detected by using Western blot analysis and fluorescent immunostaining. We examined the functional relevance of IL-33 protein in platelets by comparing platelet-intact and platelet-depleted groups in a murine model of IL-33-dependent airway eosinophilia elicited by intranasal administration of papain. We further compared the additive effect of administration of platelets derived from wild-type versus IL-33-deficient mice on the papain-induced eosinophilia. Platelets and their progenitor cells, megakaryocytes, constitutively expressed IL-33 protein (31kDa). Papain-induced IL-33-dependent airway eosinophilia in mice was significantly attenuated by platelet depletion. Conversely, concomitant administration of platelets derived from wild-type mice but not IL-33-deficient mice enhanced the papain-induced airway eosinophilia. Our novel findings suggest that platelets might be important cellular sources of IL-33 protein invivo and that platelet-derived IL-33 might play a role in airway inflammation. Therefore platelets might become an attractive novel therapeutic target for asthma and probably allergic inflammation.

Discovery of 2-((3-Amino-4-methylphenyl)amino)-N-(2-methyl-5-(3-(trifluoromethyl)benzamido)phenyl)-4-(methylamino)pyrimidine-5-carboxamide (CHMFL-ABL-053) as a Potent, Selective, and Orally Available BCR-ABL/SRC/p38 Kinase Inhibitor for Chronic Myeloid Leukemia.

Starting from a dihydropyrimidopyrimidine core scaffold based compound 27 (GNF-7), we discovered a highly potent (ABL1: IC50 of 70 nM) and selective (S score (1) = 0.02) BCR-ABL inhibitor 18a (CHMFL-ABL-053). Compound 18a did not exhibit apparent inhibitory activity against c-KIT kinase, which is the common target of currently clinically used BCR-ABL inhibitors. Through significant suppression of the BCR-ABL autophosphorylation (EC50 about 100 nM) and downstream mediators such as STAT5, Crkl, and ERK's phosphorylation, 18a inhibited the proliferation of CML cell lines K562 (GI50 = 14 nM), KU812 (GI50 = 25 nM), and MEG-01 (GI50 = 16 nM). A pharmacokinetic study revealed that 18a had over 4 h of half-life and 24% bioavailability in rats. A 50 mg/kg/day dosage treatment could almost completely suppress tumor progression in the K562 cells inoculated xenograft mouse model. As a potential useful drug candidate for CML, 18a is under extensive preclinical safety evaluation now.

Thrombin Maybe Plays an Important Role in MK Differentiation into Platelets.

Objectives. After development and differentiation, megakaryocytes (MKs) can produce platelets. As is well known, thrombopoietin (TPO) can induce MKs to differentiate. The effect of thrombin on MKs differentiation is not clear. In this study, we used a human megakaryoblastic leukemia cell line (Meg-01) to assess the effect of thrombin on MKs differentiation. Methods. In order to interrogate the role of thrombin in Meg-01 cells differentiation, the changes of morphology, cellular function, and expression of diverse factors were analyzed. Results. The results show that thrombin suppresses Meg-01 cells proliferation and induces apoptosis and cell cycle arrest. Thrombin upregulates the expression of CD41b, which is one of the most important MK markers. Globin transcription factor 1 (GATA-1), an important transcriptional regulator, controls MK development and maturation. The expression of GATA-1 is also upregulated by thrombin in Meg-01 cells. The expression of B-cell lymphoma 2 (Bcl-2), an apoptosis-inhibitory protein, is downregulated by thrombin. Phosphorylated protein kinase B (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) were upregulated by thrombin in Meg-01 cells. All the results are consistent with Meg-01 cells treated with TPO. Discussion and Conclusion. In conclusion, all these data indicate that thrombin maybe plays an important role in MK differentiation into platelets. However, whether the platelet-like particles are certainly platelets remains unknown.

Functionally Distinct Cell Populations Characterize Cord Blood Derived Megakaryocytes

A fundamental understanding of platelet biology requires functional assessment of megakaryocyte-platelet expressed genes and transcripts. The need for gene functional studies has recently increased with unbiased genome wide associations with platelet phenotypes. Animal models with an over-expressed or knocked-out gene have been particularly valuable systems, although species differences may preclude optimal evaluation. Assessing protein function in human platelets can be accomplished with pharmacologic inhibitors, although inhibitor availability and specificity can plague this approach. Studies with primary human megakaryocytes (MKs) are greatly challenged by inaccessibility, low yields and contamination with other cells. Cultured human megakaryocyte-like cell lines (e.g., Meg-01, HEL and others) have proven valuable, but these cells poorly recapitulate platelet physiology. Increasingly, investigators are using cultured human MKs to study gene function. CD34+ hematopoietic stem cells from human cord blood are routinely used to generate megakaryocytes in culture. This system has advantages of being human, relatively easy to obtain, cultured from primary cells and able to be genetically manipulated. Although cord blood-derived megakaryocytes (CBDMs) have proven useful for studying aspects of megakaryocyte/platelet biology, there is little information about the heterogeneous cell populations observed in CBDMs. In the course of our studies with CBDMs we observed multiple distinct cell populations by flow cytometry. Similar populations were also observed in the megakaryocytic Meg-01 cell line. The largest cells (called P1) were the most abundant (consisting of nearly 100% of cells) at day 3 in culture. P2 cells, which are smaller and more granular than P1, appeared at day 6 and by day 13 were ~50% of the total. P3 appeared at day 6 and are the smallest, with size and granularity roughly similar to platelets; by day 13 these were ~30% of the total. P1 but not P2 nor P3 became CD61/CD41/CD42 positive and CD34 negative over 13 days in culture. Ninety-seven percent and 93% of P2 and P3 cells, respectively, were phosphatidyl serine (PS) positive, whereas 93% of P1 cells were PS negative. Electron microscopy revealed many typical features of bone marrow MKs in the PS negative (P1) cells, including large size, polyploid nucleus, mitochondria and immature granules. However, the demarcation membrane system was poorly developed. Virtually all of the PS positive (P2) cells were apoptotic, lacked granules and had no discernable nuclei. Purification of P1 and P2 populations followed by re-culturing revealed that P1 gives rise to both the P2 and P3 populations, whereas P2 gave rise to no other population. CD34+ cells cultured with the pan-caspase inhibitor Z-VAD-FMK developed a much smaller proportion of P2 cells with a corresponding increase in P1 cells. Stimulation of these 3 populations with collagen related peptide, thrombin, protease activated receptor 1-activating peptide (PAR1-AP) and PAR4-AP showed strong integrin activation in P1 cells, but not in P2 nor P3 cells. To assess which population was susceptible to genetic manipulation, day 3 cultures were infected with a lentiviral construct containing a shRNA to talin. Talin knockdown prevented agonist-induced integrin activation in P1 cells and as expected, had no effect on the P2 or P3 population. In summary, these data indicate that only a portion of CBDMs are functional and only the P1 population should be used to assess MK gene function. Over time, the majority of CBDMs become apoptotic and the smaller P3, platelet-like particles have minimal response to agonist compared to peripheral blood platelets. These results may partially explain the difficulties in generating functional platelets in vitro and are consistent with the need to restrain apoptosis for successful megakaryocytopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Abstract 11593: Thrombus Formation Under Flow is Inhibited by the Mechanosensitive Cation Channel Blockers GsMTx-4 Peptide and Gadolinium Chloride

Introduction: Ion channels contribute significantly to platelet physiology, including control of Ca 2+ as a second messenger. Although there is a clear link between platelet activation and shear stress, no study has examined the functional relevance of mechanosensitive (MS) ion channels in platelets or their precursors, megakaryocytes. Hypothesis: Human platelets express MS cation channels such as Piezo1, which contribute to platelet function and the development of thrombosis. Methods: Thrombus formation and [Ca 2+ ] i (intracellular Ca 2+ ) responses were studied by confocal microscopy using the indicators DiOC 6 and Fluo-3, respectively, under physiological levels of shear stress in a parallel-plate flow chamber. Aggregation and [Ca 2+ ] i responses to collagen and thrombin in washed human platelets were assessed by light transmission aggregometry and Fura-2 ratiometric measurements. GsMTx-4 (2.5μM) and gadolinium chloride (GdCl 3 ) (30μM) were used as MS cation channel inhibitors, targeting Piezo1 and TRPC6 in platelets. Piezo1 mRNA and protein expression were studied in platelets and the megakaryocytic cell line Meg-01. Results: Both inhibitors significantly inhibited thrombus formation on a collagen surface under normal arterial shear rate (average thrombus volume was reduced from 48132.7±7739.4μm 3 to 13478.9±2744.8μm 3 by GdCl 3 (n=6, P&lt;0.01) and to 25125.9±6433.5μm 3 (n=6, P&lt;0.05) by GsMTx-4). GsMTx-4 also caused a reduction in thrombin-induced platelet aggregation (to 53.6±14.0% of control, n=4, P&lt;0.05), however GdCl 3 did not affect platelet shape change or aggregation under static conditions. Pre-treatment with either GsMTx-4 or GdCl 3 did not reduce calcium influx in response to TRPC6 stimulation with 60μM OAG, suggesting a MS role for Piezo1, which was found at the mRNA transcript and protein level in platelets and Meg-01 cells. Meg-01 cells showed shear rate-dependent increases in [Ca 2+ ] i levels; at a shear rate of 3989.2s -1 , F/F 0 increased to 1.38±0.05 from 1.00±0.00 baseline (n=53 cells, P&lt;0.0001), a response which was diminished by GsMTx-4, and by chelation of extracellular Ca 2+ by EGTA. Conclusions: We provide evidence for the first time for a functional contribution of MS cation channels in platelet physiology and thrombosis.