Human Megakaryocytic Cell Line Research Articles

Design and fabrication of dysprosium impregnated polyvinyl alcohol hydrogels. Physiochemical, mechanical, bioimaging and in vitro evaluation

Tissue engineering has gained prominence during the past decade since it offers a key solution to defects associated with the tissue regeneration. The limited healing potential of the cartilage tissue damage has significant clinical implications. Herein, dysprosium (Dy3+) impregnated polyvinyl alcohol (PVA) hydrogels have been developed to enhance the therapeutic efficacy, enabling simultaneous diagnostic imaging and antibacterial drug delivery for potential applications in articular cartilage. Based on the favorable imaging features, Dy3+ impregnated PVA hydrogels with enhanced stability were formed through successive steps of repeated cycles of freezing at – 30 °C for 21 h, thawing at 25 °C for 4 h and lyophilization. The tensile and compression tests of the hydrogels respectively determined a maximum of 3.88 and 1.58 MPa, which reflected better compatibility towards cartilage. The hydrogels fetched a sustained drug release for a period of 12 h with an associated swelling ratio of 80%. The potential of the resultant hydrogels in image diagnosis has been deliberated through their blue and yellow emissions in the visible region. Further, the computed tomography (CT) and magnetic resonance imaging characteristics of the hydrogels respectively accomplished a maximum of 343 Hounsfiled units (HU) and relaxivity of 7.25 mM−1s−1. The cytocompatibility of the hydrogels is also determined through in vitro tests performed in Murine pro B cell line (BA/F3) and human Megakaryocyte cell line (Mo7e) cell lines.

Improved intravenous lentiviral gene therapy based on endothelial-specific promoter-driven factor VIII expression for hemophilia A

BackgroundHemophilia A (HA) is an X-linked monogenic disorder caused by deficiency of the factor VIII (FVIII) gene in the intrinsic coagulation cascade. The current protein replacement therapy (PRT) of HA has many limitations including short term effectiveness, high cost, and life-time treatment requirement. Gene therapy has become a promising treatment for HA. Orthotopic functional FVIII biosynthesis is critical to its coagulation activities.MethodsTo investigate targeted FVIII expression, we developed a series of advanced lentiviral vectors (LVs) carrying either a universal promoter (EF1α) or a variety of tissue-specific promoters, including endothelial-specific (VEC), endothelial and epithelial-specific (KDR), and megakaryocyte-specific (Gp and ITGA) promoters.ResultsTo examine tissue specificity, the expression of a B-domain deleted human F8 (F8BDD) gene was tested in human endothelial and megakaryocytic cell lines. Functional assays demonstrated FVIII activities of LV-VEC-F8BDD and LV-ITGA-F8BDD in the therapeutic range in transduced endothelial and megakaryocytic cells, respectively. In F8 knockout mice (F8 KO mice, F8null mice), intravenous (iv) injection of LVs illustrated different degrees of phenotypic correction as well as anti-FVIII immune response for the different vectors. The iv delivery of LV-VEC-F8BDD and LV-Gp-F8BDD achieved 80% and 15% therapeutic FVIII activities over 180 days, respectively. Different from the other LV constructs, the LV-VEC-F8BDD displayed a low FVIII inhibitory response in the treated F8null mice.ConclusionsThe LV-VEC-F8BDD exhibited high LV packaging and delivery efficiencies, with endothelial specificity and low immunogenicity in the F8null mice, thus has a great potential for clinical applications.

Abstract LB256: Type 1 calreticulin mutations in myeloproliferative neoplasms promote glycolysis via intracellular calcium mediated upregulation of GLUT1

Abstract Myeloproliferative neoplasms (MPNs) comprise a group of cancers of the bone marrow that can transform into a post-MPN acute myeloid leukemia (AML), at which point the overall survival rate is less than 6 months. JAK2 inhibitors in MPN failed to alter disease progression, emphasizing the need for new treatments. Calreticulin (CALR) is one of the MPN driver mutations and encodes an endoplasmic reticulum (ER) calcium (Ca2+) binding protein. CALR mutations are classified as either type 1 or type 2. Only type 1 proteins exhibit loss of the C-terminal Ca2+ binding sites. Additionally, type 1 and 2 CALR mutations engender significant prognostic differences. Previously published work from our lab showed that type 1 CALR exhibits a loss of Ca2+ binding sites, rendering them unable to bind ER Ca2+ and leading to depletion of ER Ca2+ stores. We next wanted to evaluate where this Ca2+ is localized. Through imaging studies, we found that CALR type 1 expressing cells have significantly increased cytosolic Ca2+ and significantly lower mitochondrial Ca2+ compared to both CALR wildtype and CALR type 2 expressing cells. It has been well established in various cancer types that increased glycolytic activity is prompted in part by elevated cytosolic Ca2+. To test if this was the case in our model, we performed a vast array of metabolic assays in UT-7-MPL cells, a human megakaryocytic cell line, including but not limited to lactate secretion, Seahorse XF Glycolysis Stress Test assay, and metabolite tracing with 13-C labelled glucose. These all confirmed a highly glycolytic profile in UT-7-MPL cells expressing the CALR type 1 mutation. In addition, through evaluation of mitochondrial mass, membrane potential and several other assays, mitochondrial metabolism in CALR type 1 mutant cells appears severely dysfunctional. All the glycolytic and mitochondrial phenotypes are almost fully rescued upon the reintroduction of the Ca2+ binding sites of the CALRwt protein (P and C domains; P+C), indicating that perturbed Ca2+ homeostasis is the driver of these changes. Pharmacological treatment with cytosolic Ca2+ chelator BAPTA-AM or with kaempferol, an activator of the mitochondrial Ca2+ uniporter mimic the P+C rescue effect for both the glycolytic and the mitochondrial phenotypes. In our in vitro system CALR type 1 mutant cells express along with other glycolytic factors significantly higher cell surface levels of the glucose transporter GLUT1, and demonstrate enhanced sensitivity to pharmacological GLUT1 inhibition with BAY-876. Preliminary data in a transgenic mouse model as well as in primary patient samples show that GLUT1 expression is higher in CALR type 1 mutant mice and patient cells, and that the mitochondrial mass in this subset of patients is lower compared to CALR type 2 mutant patients. Finally, we validated that all these phenotypes are JAK/STAT pathway independent and that they are worsened by ruxolitinib treatment, providing a strong rationale for a combination treatment. Together, these data both expand the current knowledge of the role that Ca2+ dynamics play in metabolic reprogramming and can lead to improved targeted therapies for MPN patients. Citation Format: Michele Ciboddo, Jonathan Dowgielewicz, George Yan, Deborah Rodriguez, Hunter Blaylock, Nicole Arellano, Chad Coen, Chufan Cai, Nia Hammond, Alex Rosencrance, Elisa Rumi, Daniela Pietra, Daniele Vanni, Silvia Catricalà, Ilaria Carola Casetti, Oscar Borsani, Brandon Faubert, Shannon Elf. Type 1 calreticulin mutations in myeloproliferative neoplasms promote glycolysis via intracellular calcium mediated upregulation of GLUT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB256.

Identification and characterization of bioactive metabolites of 12-hydroxyheptadecatrienoic acid, a ligand for leukotriene B4 receptor 2.

12(S)-hydroxyheptadecatrienoic acid (12-HHT) is a bioactive fatty acid synthesized from arachidonic acid via the cyclooxygenase pathway and serves as an endogenous ligand for the low-affinity leukotriene B4 receptor 2 (BLT2). Although the 12-HHT/BLT2 axis contributes to the maintenance of epithelial homeostasis, 12-HHT metabolism under physiological conditions is unclear. In this study, 12-keto-heptadecatrienoic acid (12-KHT) and 10,11-dihydro-12-KHT (10,11dh-12-KHT) were detected as 12-HHT metabolites in the human megakaryocytic cell line MEG01s. We found that 12-KHT and 10,11dh-12-KHT are produced from 12-HHT by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and prostaglandin reductase 1 (PTGR1), key enzymes in the degradation of prostaglandins, respectively. The 15-PGDH inhibitor SW033291 completely suppressed the production of 12-KHT and 10,11dh-12-KHT in MEG01s cells, resulting in a 9-fold accumulation of 12-HHT. 12-KHT and 10,11dh-12-KHT were produced in mouse skin wounds, and the levels were significantly suppressed by SW033291. Surprisingly, the agonistic activities of 12-KHT and 10,11dh-12-KHT on BLT2 were comparable to that of 12-HHT. Taken together, 12-HHT is metabolized into 12-KHT by 15-PGDH, and then 10,11dh-12-KHT by PTGR1 without losing the agonistic activity.

Apoptosis of Megakaryocytic Leukemia Cell Line Meg-01 Induced by TSP-1 Via CD36/Caspase-3

To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. 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. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 μg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 μg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 μg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 μg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 μg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while β-TG could not. CD36 antibody FA6-152 (25 μg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic 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.

Intravenous Lentiviral Gene Therapy for Hemophilia a: In Vitro and In Vivo Assessment of Universal Versus Tissue-Specific Promoters

Background：Hemophilia A (HA) is an X-linked monogenic coagulation disorder resulting from deficiency of the factor VIII (FVIII, F8) gene in the intrinsic coagulation cascade. The current treatment of HA is based on protein replacement therapy (PRT) through plasma-derived coagulation factors or recombinant proteins with limitations of short half-life, high cost, and life-time requirement of the treatment. Gene therapy has become a promising treatment for HA.Methods and Materials: We developed an advanced lentiviral vector (LV) system for intravenous (iv) F8 gene therapy. A selective codon optimized and B-domain deleted human F8 (hF8BDD) gene was synthesized, sequenced and functionally verified. LVs carrying a universal EF1α promoter, or several modified tissue-specific promoters including endothelial- (VEC), endothelial and epithelial- (KDR), and two megakaryocyte-specific (ITGA and Gp) promoters, were biologically and immunologically characterized in vitro using human endothelial and megakaryocytic cell lines, EA-hy926 and DAMI, and in vivo using F8 knockout (KO) mice.Results: We investigated the different LV promoter constructs and found that LV-VEC-F8BDD exhibited the highest virus packaging (1.3x10 9 transduction units/ml) and transduction efficiencies compared with the other LV constructs. Tissue-specific expression of the VEC, KDR, ITGA and Gp promoters was confirmed in EA-hy926 and DAMI cells by RT-PCR, Western blot and ELISA analyses. We detected F8 activities close to 6 folds and 4.5 folds above the normal plasma level from the EF1a LV-F8BDD transduced EA-hy926 cells and DAMI cells, respectively, whereas the VEC LV in EA-hy926 cells and the ITGA-LV in DAMI cells exhibited F8 activities at 1.5 folds and 5 folds above the normal plasma level, respectively. In vivo studies in F8 KO mice via iv injection of LVs after reduced radiation conditioning illustrated preferential vector expression in different cell lineages, with high expression of EF1a vector in CD11b, F4/80 and Ly-6G positive immune cells, and preferential expression of VEC vector in CD31 positive endothelial cells, and ITGA and Gp vectors in CD41 positive megakaryocytes. In addition, we detected variable phenotypic corrections as well as anti-F8 immune responses in the F8 KO mice treated with the different LVs. The iv deliveries of VEC and Gp F8BDD vectors illustrated therapeutic F8 activities over time, around 25% and 8%, respectively, in 60 days, which increased to high levels (80% and 25%, respectively) after 120 days (Figure A). Kinetic analyses of anti-F8 IgG and inhibitor titers (Bethesda assay) of the treated mice showed that the VEC vector exhibited the lowest F8 inhibitory immune response over time (Figure B).Conclusion: Based on the in vitro and in vivo studies, our results suggest that for HA gene therapy, optimal rather than high F8 expression is critical, and tissue-specific expression but not universal expression can reduce adverse inhibitor effect. We demonstrated that the LV-VEC-F8BDD vector displayed high tissue specificity in vivo, and high transgene delivery efficiency, high coagulation function and low immunogenicity. In addition, iv LV gene therapy could be a safe, convenient and effective HA gene therapy strategy. [Display omitted] 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.

Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression

Platelets are generated from the cytoplasm of megakaryocytes (MKs) via actin cytoskeleton reorganization. Zyxin is a focal adhesion protein and wildly expressed in eukaryotes to regulate actin remodeling. Zyxin is upregulated during megakaryocytic differentiation; however, the role of zyxin in thrombopoiesis is unknown. Here we show that zyxin ablation results in profound macrothrombocytopenia. Platelet lifespan and thrombopoietin level were comparable between wild-type and zyxin-deficient mice, but MK maturation, demarcation membrane system formation, and proplatelet generation were obviously impaired in the absence of zyxin. Differential proteomic analysis of proteins associated with macrothrombocytopenia revealed that glycoprotein (GP) Ib-IX was significantly reduced in zyxin-deficient platelets. Moreover, GPIb-IX surface level was decreased in zyxin-deficient MKs. Knockdown of zyxin in a human megakaryocytic cell line resulted in GPIbα degradation by lysosomes leading to the reduction of GPIb-IX surface level. We further found that zyxin was colocalized with vasodilator-stimulated phosphoprotein (VASP), and loss of zyxin caused diffuse distribution of VASP and actin cytoskeleton disorganization in both platelets and MKs. Reconstitution of zyxin with VASP binding site in zyxin-deficient hematopoietic progenitor cell-derived MKs restored GPIb-IX surface expression and proplatelet generation. Taken together, our findings identify zyxin as a regulator of platelet biogenesis and GPIb-IX surface expression through VASP-mediated cytoskeleton reorganization, suggesting possible pathogenesis of macrothrombocytopenia.

Activation Mediated By Peroxisome Proliferator-Activated Receptor (PPAR) Ligands and Brown Algae-Based Compounds: Different Mechanisms between Megakaryoblasts and Mature Platelets

Background: Nuclear receptors aretranscription factors that are activated by ligands and subsequently bind to regulatory regions in target genes. Recently ligand-activated transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) has been reported to promote the formation of platelets from megakaryocytes and accelerate platelet recovery after radiation-induced bone marrow injuries. The endogenous prostaglandin 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2) are ligands of PPARγ. Accumulating evidences also suggest that PPAR activation is involved in inflammation responses and atherosclerosis. In the present study, we analyzed how PPAR ligands and some brown algae-based compounds affect platelet differentiation and functions in vitro.Methods: Platelets were obtained from plateletpheresis products by centrifugation. DAMI and Meg-1 cells, both human megakaryocytic cell lines, were also obtained. Western blotting for PPARγ, PPARα and COX-2 was performed, and bands were visualized by chemiluminescence. The effects of 15d-PGJ2, GW9662, Fucoidan, Seanol, GW6471 and Celecoxib were evaluated. Morphology analysis, platelet flow cytometry, total thrombus formation analysis system (T-TAS) and microarray expression analysis were also used in order to analyze the effects of the treatment on platelets.Results: Megakaryoblasts and mature platelets showed different results in the expression of PPARα, COX-2, glycoproteins and platelet activation markers. PGJ2 induced morphological differentiation evidences in DAMI and Meg-1 cells. PGJ2 increased the levels of CD41, CD42b, CD62p and PAC-1 both in DAMI and Meg-1 cells. In mature platelets, Fucoidan and Seanol decreased the expression of PPARα and increased COX-2 expression. Fucoidan elevated the expression of CD62p and CD63 in mature platelets, suggesting that Fucoidan causes platelet activation. In microarray analysis, fucoidan showed early growth response (EGR)-1 gene upregulation. Unfortunately thrombogenesity by T-TAS was not evident from the study.Conclusions: The mechanism of platelet production and activation is not fully understood. Understanding the mechanisms underlying megakaryocyte maturation and platelet production will facilitate the development of methods to increase the production of platelets from precursor cells. Although platelets are non-nucleated cells, they contain transcription factors, especially PPAR and NF-κB. From the present study, our findings support the hypothesis that PPAR, both PPARγ and PPARα play a role in platelet differentiation and activation, and exact mechanisms need to be defined related to prostaglandin pathways. Fucoidan may activate platelets via different pathways than PGJ2. DisclosuresNo relevant conflicts of interest to declare.

Synthesis of Inflammatory Cytokines and Expression of Adhesion Molecules in Megakaryocytes

Inflammatory cytokines and platelet adhesion molecules may play a major role in hemostasis and immuno-pathological processes. Hematopoietic cells of myeloid and lymphoid lineages are known to be important sources of a variety of inflammatory cytokines. However, the immunological role of megakaryocytes is not well known. This project studied the involvement of megakaryocytes on immuno-inflammatory processes and the possible mechanism via the adhesion molecules and the synthesis of relevant cytokines. The expression of interleukin (IL)-1 to IL-10, TNF-a, TNF-g and IFN-g in primary megakaryocytes and four megakaryocytic cell lines: Meg-01, Dami, CHRF-288-11 and M-07e were determined by RT-PCR. The effect of IL-1b, IL-3, IL-6 and TPO on megakaryocyte colony formation (CFU-MK) was studied by using a plasma clot culture system. The CFU-MK was confirmed by AchE staining. The expression of adhesion protein CD36 on human platelets, megakaryocytes and megakaryocytic cell lines was also analyzed by using flow cytometry, ELISA and immunocytochemical methods. Our results showed that CD36 was expressed on platelets, megakaryocytes and the four megakaryocytic cell lines. The relative expression level is as follows: platelets > megakaryocytes > Meg-01 > Dami > CHRF-288-11 > M-07e, suggesting that the level of CD36 expression correlates with the degree of maturity of megakaryocytic differentiation. Inflammatory cytokines TNF-a, IL-1b, IL-3 and IL-6 were detected in primary megakaryocytes and all the four megakaryocytic cell lines, suggesting that different stages of megakaryocytes can be as a source of inflammatory cytokines. IL-1b, IL-3 and IL-6, as well as TPO, play a stimulating effect on CFU-MK formation, suggesting that there is an “autocrine” effect on megakaryopoiesis. Megakaryocytes may involve in immuno-inflammatory processes via the synthesis of inflammatory cytokines and platelet adhesion molecules. DisclosuresNo relevant conflicts of interest to declare.

Anti-Glucosylsphingosine Autoimmunity, JAK2V617F-Dependent Interleukin-1β and JAK2V617F-Independent Cytokines in Myeloproliferative Neoplasms.

Simple SummaryInflammation plays a major role in myeloproliferative neoplasms (MPNs) as regulator of malignant cell growth and mediator of clinical symptoms. Yet chronic inflammation may also be an early event that facilitates the development of MPNs. Here we analysed 42 inflammatory cytokines and report that in patients as well as in UT-7 cell lines, interleukin-1β and interferon-induced protein 10 (IP-10) were the main inflammatory molecules found to be induced by JAK2V617F, the most frequent driving mutation in MPNs. All other inflammatory cytokines were not linked to JAK2V617F, which implies that inflammation likely precedes MPN development at least in subsets of MPN patients. Consistently, a possible cause of early, chronic inflammation may be auto-immunity against glucolipids: we report that 20% of MPN patients presented with anti-glucosylsphingoside auto-antibodies. Since existing treatments can reduce glucosylsphingoside, this lysosphingolipid could become a new therapeutic target for subsets of MPN patients, in addition to JAK2V617F and inflammation.Inflammatory cytokines play a major role in myeloproliferative neoplasms (MPNs) as regulators of the MPN clone and as mediators of clinical symptoms and complications. Firstly, we investigated the effect of JAK2V617F on 42 molecules linked to inflammation. For JAK2V617F-mutated patients, the JAK2V617F allele burden (%JAK2V617F) correlated with the levels of IL-1β, IL-1Rα, IP-10 and leptin in polycythemia vera (PV), and with IL-33 in ET; for all other molecules, no correlation was found. Cytokine production was also studied in the human megakaryocytic cell line UT-7. Wild-type UT-7 cells secreted 27/42 cytokines measured. UT-7 clones expressing 50% or 75% JAK2V617F were generated, in which the production of IL-1β, IP-10 and RANTES was increased; other cytokines were not affected. Secondly, we searched for causes of chronic inflammation in MPNs other than driver mutations. Since antigen-driven selection is increasingly implicated in the pathogenesis of blood malignancies, we investigated whether proinflammatory glucosylsphingosine (GlcSph) may play a role in MPNs. We report that 20% (15/75) of MPN patients presented with anti-GlcSph IgGs, distinguished by elevated levels of 11 cytokines. In summary, only IL-1β and IP-10 were linked to JAK2V617F both in patients and in UT-7 cells; other inflammation-linked cytokines in excess in MPNs were not. For subsets of MPN patients, a possible cause of inflammation may be auto-immunity against glucolipids.

The balance of life and death may hinge on the intracellular localization of the small GTPase, Rap1b in the megakaryocyte cell line (DAMI)

Platelets are specialized cell fragments that circulate in the bloodstream and clot to prevent blood from leaving damaged vessels. The number of circulating platelets is governed by the balance of platelet production by mature megakaryocytes and clearance of platelets from the bloodstream. Normal platelet function requires the signaling protein, Rap1b, whose abundance also increases as megakaryocytes mature. The intracellular location of Rap1b is determine by its phosphorylation state. When Rap1b is not phosphorylated, it associates with membranes; conversely when Rap1b is phosphorylated, it detaches from membranes and reside in the cytosol. We hypothesize that Rap1b, specifically in its phosphorylated, cytosolic state, promotes megakaryocytes survival and proliferation. To address this hypothesis, the human megakaryocyte cell line (DAMI cells) have been modified 1) by disrupt the rap1b gene creating a Rap1b‐knockout cell line, 2) stable overexpression of a Rap1b phospho‐mimetic mutant, 3) stable overexpression of a Rap1b phospho‐deficient mutant or 4) stable overexpression of wild‐type Rap1b (to be used as a control). Using the modified DAMI cell lines we are beginning to understand the impact of Rap1b phosphorylation on proliferation on in MTS survival assays. Changes in cell survival can be attributed to changes in cell cycle progression and/or susceptibility to apoptosis. To address possible changes in cell cycle progression, we have evaluated each cell line based on the percentage of cell undergoing DNA synthesis at a given time by BrdU incorporation assays. To evaluate apoptosis susceptibility, mitochondrial membrane integrity and activation/cleavage of Caspase 9 was determined in the presence or absence of the apoptotic inducer ABT‐737. The analysis of the role of Rap1b in megakaryocytes has the potential to increase our collective understanding of regulation of platelet count and could be a novel target to combat thrombocytopenia (low platelet count) or thrombocythemia (high platelet count).Support or Funding InformationUniversity of Wisconsin ‐LaCrosse Faculty Research Grant

Dengue viruses infect human megakaryocytes, with probable clinical consequences.

One of the most important clinical signs of dengue virus infection is the reduction of white blood cells and platelets in human peripheral blood (leukopenia and thrombocytopenia, respectively), which may significantly impair the clearance of dengue virus by the immune system. The cause of thrombocytopenia and leukopenia during dengue infection is still unknown, but may be related to severe suppression of bone marrow populations including hematopoietic stem cells and megakaryocytes, the progenitors of white blood cells and platelets respectively. Here, we explored the possibility that bone marrow suppression, including ablation of megakaryocyte populations, is caused by dengue virus infection of megakaryocytes. We used three different models to measure dengue virus infection and replication: in vitro, in a human megakaryocyte cell line with viral receptors, ex vivo, in primary human megakaryocytes, and in vivo, in humanized mice. All three systems support dengue virus infection and replication, including virus strains from serotypes 1, 2, and 3, and clinical signs, in vivo; all assays showed viral RNA and/or infectious viruses 7–14 days post-infection. Although we saw no significant decrease in cell viability in vitro, there was significant depletion of mature megakaryocytes in vivo. We conclude that megakaryocytes can produce dengue viruses in the bone marrow niche, and a reduction of cell numbers may affect bone marrow homeostasis.

Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia

AbstractInherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

Dynamins 2 and 3 Are Required for Human Megakaryocytes Directional Migration

Abstract:Megakaryocytes (MKs) undergo directional migration from the proliferative osteoblastic niche within the bone marrow (BM) environment to the capillary-rich vascular niche for platelet production and release into the pulmonary circulation. This process is regulated, in part by dynamins, large GTPase proteins that regulate cellular functions such as endocytosis, vesicle transport and cell migration. Additional functions of dynamins include the formation of actin-rich structures, such as lamellipodia and dorsal membrane ruffles, invadopodia and podosomes. Previous studies have shown that mutations in Dynamin 2 (DNM2) cause thrombocytopenia in humans. To explore the function of dynamins in megakaryocyte migration and platelet production in more depth, we monitored the response of cells to chemotaxis SDF1α gradient signal by a microfluidic device-based approach. We observed an impaired directional migration by both human megakaryocytic cell lines and primary cells treated either with dynasore, a small molecule inhibitor of dynamins, or shRNA knockdown of Dynamin 2 and 3 (DNM2, DNM3). Since directional cell migration is tightly regulated by actin cytoskeletal rearrangements, we next measured actin polymerization and RhoA activity. We observed a profound decrease in the F-actin and Rho GTPase activity upon loss of DNM2 and DNM3 function. Next, since the response to chemoattractant signal is navigated by SDF1 through its receptor CXCR4, we explored the CXCR4 receptor response to ligand in dynamin defective megakaryocytes. Interestingly we observed an increase in CXCR4 expression in the dynasore treated primary human cells. Additionally, combined inhibition of DNM2 and DNM3 or over expression of dominant negative Dnm2-K44A or GTPase-defective DNM3 decreased the active β1- integrin (ITGB1) activity, which indicates a decrease in the integrin mediated endo/exocytic cycling during cell migration. Finally, to understand the role of dynamin in endosome recycling, we assayed the distribution of Rab11, a marker of recycling endosomes. We noticed an abnormal clustered staining pattern of Rab11 in dynasore-treated MKs which is indicative of a disruption in recycling pathways. This observation suggests decreased recruitment of the recycling pathway in dynasore-treated cells. Altogether, in this study we demonstrate that dynamins regulate MKs directional migration towards the SDF1α chemotaxis signal in the bone marrow and governs endocytosis and cell receptor trafficking. DisclosuresCrispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Anaplasma phagocytophilum infection modulates expression of megakaryocyte cell cycle genes through phosphatidylinositol-3-kinase signaling

Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis infects neutrophils and other cells from hematopoietic origin. Using human megakaryocytic cell line, MEG-01, we show that expression of cell cycle genes in these cells are altered upon A. phagocytophilum infection. Expression of several cell cycle genes in MEG-01 cells was significantly up regulated at early and then down regulated at later stages of A. phagocytophilum infection. Lactate dehydrogenase (LDH) assays revealed reduced cellular cytotoxicity in MEG-01 cells upon A. phagocytophilum infection. The levels of both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and inhibitory kappa B (IκB) were elevated at both early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in significant reduction in the expression of tested cell cycle genes, A. phagocytophilum burden and phosphorylated Akt levels in these MEG-01 cells. Collectively, these results suggest a role for PI3K-Akt-NF-κB signaling pathway in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection.

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.

Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis.

Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)-targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r(-/-)), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r(-/-) mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events.

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

SummaryIon channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288–11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These “channelome” datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.