Number Of Megakaryocytes Research Articles

The study of the protective properties of vitamin E on bone marrow of rats in condition of the increased intake of heavy metals salts

Homeostasis imbalance leads to the changes in qualitative and quantitative characteristics of all hematopoietic sprouts. It encourages to search for the effective protective means in the conditions of the polluted environment. Our research is focused on the study the protective properties of vitamin E on the bone marrow of the rats in conditions of the increased intake of the heavy metals salts. During the research the bone marrow of the 36 laboratory male rats was studied. The sections of the decalcified femoral bones were stained using hematoxylin and eosin method, Van Gieson’s technique and PAS-reaction, as well as immunohistochemical study to CD3, CD79α, S100 and MРО was used. Elemental composition of hematopoietic tissue was determined through atomic spectrophotometry. The accumulation of the heavy metals led to microelementosis (changes in qualitative and quantitative characteristics of all hematopoietic sprouts and surrounding tissues). The extension of the influence of the exogenous pollutants led to the suppression of erythro-and leucopoiesis, synchronous increase in the number of megakaryocytes. These indicators reached the significant deviations on the 90 day of the observation. The use of the vitamin E as a corrector reduces the negative effect of the pollutants that is demonstrated by the reduced accumulation of the heavy metals in the bone marrow, prevention of the significant deviations in the quantitative and qualitative composition of hematopoietic tissue.

Substantial improvement in immune thrombocytopenic purpura associated with T-cell/histiocyte-rich B-cell lymphoma treated with chemotherapy: A case report.

Immune thrombocytopenic purpura (ITP) is characterized by antibody and immune platelet destruction, occasionally leading to life-threatening hemorrhage. ITP is a frequent complication of chronic lymphoid leukemia, but is rarely associated with diffuse large B-cell lymphoma (DLBCL). We herein describe a case of ITP associated with T-cell/histiocyte-rich B-cell lymphoma (T/HRBCL), which is a rare variant of DLBCL. A 54-year-old man presented with fever, multiple lymphadenopathies, petechial purpura and intraoral mucosal hemorrhage. Laboratory data revealed thrombocytopenia, normal white blood cell count, mild anemia, no active viral infections, and no autoimmune antibodies. Multiple lymphadenopathies were observed on computed tomography. A bone marrow biopsy revealed minor hypercellularity with increased number of megakaryocytes; however, there were no pathological cells or phagocytic cells. The patient was diagnosed with ITP; concurrently, he was also diagnosed with T/HRBCL via lymph node biopsy, and was treated accordingly with a chemotherapeutic regimen comprising rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone. Prompt reduction in the size of the superficial lymph nodes and an increase of the platelet count were observed simultaneously. To the best of our knowledge, this is the first report of T/HRBCL complicated by ITP. The findings strongly suggest a causative association between ITP and T/HRBCL.

Inhibition of Tropomyosin Receptor Kinase A Signaling Negatively Regulates Megakaryopoiesis and induces Thrombopoiesis

Neurotrophin signaling modulates the differentiation and function of mature blood cells. The expression of neurotrophin receptors and ligands by hematopoietic and stromal cells of the bone marrow indicates that neurotrophins have the potential to regulate hematopoietic cell fate decisions. This study investigates the role of neurotrophins and Tropomyosin receptor kinases (Trk) in the development of megakaryocytes (MKs) and their progeny cells, platelets. Results indicate that primary human MKs and MK cells lines, DAMI, Meg-01 and MO7e express TrkA, the primary receptor for Nerve Growth Factor (NGF) signaling. Activation of TrkA by NGF enhances the expansion of human MK progenitors (MKPs) and, to some extent, MKs. Whereas, inhibition of TrkA receptor by K252a leads to a 50% reduction in the number of both MKPs and MKs and is associated with a 3-fold increase in the production of platelets. In order to further confirm the role of TrkA signaling in platelet production, TrkA deficient DAMI cells were generated using CRISPR-Cas9 technology. Comparative analysis of wild-type and TrkA-deficient Dami cells revealed that loss of TrkA signaling induced apoptosis of MKs and increased platelet production. Overall, these findings support a novel role for TrkA signaling in platelet production and highlight its potential as therapeutic target for Thrombocytopenia.

Anti-apoptotic BCL2L2 increases megakaryocyte proplatelet formation in cultures of human cord blood.

Apoptosis is a recognized limitation to generating large numbers of megakaryocytes in culture. The genes responsible have been rigorously studied in vivo in mice, but are poorly characterized in human culture systems. As CD34-positive (+) cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity (LLG), and smaller size, higher granularity (SHG). The LLG cells were CD41aHigh CD42aHigh phosphatidylserineLow, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and displayed a signaling response to platelet agonists. The SHG cells were CD41aLowCD42aLowphosphatidylserineHigh, had a distinctly apoptotic morphology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte apoptosis regulator. Lentiviral BCL2L2 overexpression decreased megakaryocyte apoptosis, increased CD41a+ LLG cells, and increased proplatelet formation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet BCL2L2 mRNA levels. This finding was consistent with the observed increase in platelet-like particles derived from cultured megakaryocytes over-expressing BCL2L2. BCL2L2 also induced small, but significant increases in thrombin-induced platelet-like particle αIIbβ3 activation and P-selectin expression. Thus, BCL2L2 restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation, and is associated with platelet number. BCL2L2 is a novel target for improving megakaryocyte and platelet yields in in vitro culture systems.

Packaging functionally important plasma proteins into the α-granules of human-induced pluripotent stem cell-derived megakaryocytes.

The contents of platelet α-granules arrive via a number of pathways; some are synthesized by megakaryocytes (MKs), for example, von Willebrand factor (VWF), whereas others are endocytosed from plasma, for example, fibrinogen (Fgn) and factor V (FV). Currently, almost all in vitro-induced pluripotent stem cell (iPSC)-derived MKs are generated under serum-free conditions, and their α-granule cargoes lack components that would normally be taken up from plasma during the course of megakaryopoiesis. How this might affect the ability of in vitro-derived platelets to contribute fully to haemostasis is not known. The purpose of this investigation was to examine whether "feeding" human plasma to iPSC-derived MKs might result in loading their α-granules with physiologically important proteins. iPSCs were differentiated to CD41+ /CD42b+ MKs using a serum-free protocol. The resulting MKs were polyploid, expressed a number of platelet-specific surface receptors, and spread on Fgn or collagen-coated surfaces. Reverse transcription-polymerase chain reaction analysis detected mRNA transcripts for FV and VWF but not Fgn chains. Fluorescence immunocytochemistry and confocal microscopy confirmed constitutive VWF distribution in granule-like structures in MKs cultured under plasma-free conditions, and the granules became positive for Fgn upon incubation with human plasma. iPSC-derived MKs showed a low level of constitutive FV expression that increased dramatically upon incubation with human plasma. Taken together, these data suggest that human iPSC-derived MKs are capable of endocytosing and storing plasma components in their α-granules. Incorporating this methodology into current protocols for producing in vitro-derived MKs should provide novel insights into MK biology and lead to the generation of large numbers of MKs and platelets with improved functionality.

Bone Marrow Morphology Associated With Germline RUNX1 Mutations in Patients With Familial Platelet Disorder With Associated Myeloid Malignancy.

Germline mutations in RUNX1 result in autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM). To characterize the hematopathologic features associated with a germline RUNX1 mutation, we reviewed a total of 42 bone marrow aspirates from 14 FPDMM patients, including 24 cases with no cytogenetic clonal abnormalities, and 18 with clonal karyotypes or leukemia. We found that all aspirate smears had ≥10% atypical megakaryocytes, predominantly characterized by small forms with hypolobated and eccentric nuclei, and forms with high nuclear-to-cytoplasmic ratios. Core biopsies showed variable cellularity and variable numbers of megakaryocytes with similar features to those in the aspirates. Granulocytic and/or erythroid dysplasia (≥10% cells per lineage) were present infrequently. Megakaryocytes with separate nuclear lobes were increased in patients with myelodysplastic syndrome (MDS) and acute leukemia. Comparison to an immune thrombocytopenic purpura cohort confirms increased megakaryocytes with hypolobated eccentric nuclei in FPDMM patients. As such, patients with FPDMM often have atypical megakaryocytes with small hypolobated and eccentric nuclei even in the absence of clonal cytogenetic abnormalities; these findings are related to the underlying RUNX1 germline mutation and not diagnostic of MDS. Isolated megakaryocytic dysplasia in patients with unexplained thrombocytopenia should raise the possibility of an underlying germline RUNX1 mutation.

Stroke, Carotid Thrombosis and Other Thrombotic Events in Essential Thrombocythemia Patient with a High Quantity of Micromegakaryocytes

Essential thrombocythemia is a myeloproliferative neoplasm characterized by thrombocytosis and possible complications such as thrombosis, hemorrhage, splenomegaly, bone marrow failure and acute leukemia. In the myeloproliferatives neoplasms, there is an increase number of dysplastic megakaryocytes, known as micromegakaryocytes and there is an association between the high expression of the JAK2V617F mutation and the presence of micromegakaryocytes in bone marrow. To date, reports and researches involving essential thrombocythemia and micromegakaryocytes are exceedingly scarce. We report a peculiar and unique case of a death of a 55-year-old man, due to stroke and carotid thrombosis, preceded by successive thrombotic events in a patient with essential thrombocythemia, JAK2 positive, with no additional risk factor, who did not respond well to standard treatment and had a high amount of circulating micromegakaryocytes in peripheral blood. This case goes beyond the known prognosis of ET and raises the discussion of new prognostic markers, such as the quantification of micromegakaryocytes and reaffirms the importance of the JAK2 mutation in the evolution of the disease. We also discuss the involvement of micromegakaryocytes in the probable mechanisms leading to our patient’s thrombotic state.

A novel recombinant human thrombopoietin for treating prolonged isolated thrombocytopenia after allogeneic stem cell transplantation

Patients with prolonged isolated thrombocytopenia (PT) after allogeneic stem cell transplantation (allo-SCT) are known to have a poor prognosis. However, there is no standard treatment for PT. The present study aimed to investigate the potential effect of a novel recombinant human thrombopoietin (rhTPO) in a cohort of patients with PT following allo-SCT. A total of 24 patients with PT (including delayed platelet engraftment and secondary failure of platelet recovery) were treated with rhTPO from July 1, 2016 to May 31, 2017. rhTPO injections were administered at 300 IU/kg/d for 28 consecutive days or until platelet counts were ≥ 50 × 109/L, independent of platelet transfusion. Response was defined as platelet recovery to ≥ 20 × 109/L for seven consecutive days without transfusion support during or within 7 days of the end of rhTPO treatment. All patients completed the 28 days of treatment, and none were withdrawn due to adverse effects. The overall response was 45.8%, which was significantly higher than historical data (12.2%, p < 0.001). The median response time was 12 (7–25) days from the initiation of rhTPO treatment. A response to rhTPO was associated with megakaryocytes in the bone marrow (positive vs. negative, 81.8 vs. 22.2%; p = 0.022). Among 11 patients exhibiting a response to rhTPO, the median number of megakaryocytes in bone marrow was increased significantly (10 vs. 2; p = 0.036) after rhTPO treatment. In conclusion, the results of this preliminary study suggest that rhTPO may represent a therapeutic option, with a response of 45.8% for patients with PT after allo-SCT, and especially for those with megakaryocytes in the bone marrow. However, this should be further confirmed in randomized prospective clinical trials.

αIIb(R990W), a Gain-of Function Mutation of αIIbβ3, Knock-in Mice Show Moderately Impaired Thrombopoiesis

Introduction:Integrin αIIbβ3 plays an essential role in platelet aggregation, and quantitative or qualitative defects in αIIbβ3 lead to severe bleeding diathesis, which is known as Glanzmann thrombasthenia (GT). Although platelet counts and morphology are usually normal in GT patients, recent several reports demonstrate that constitutive gain-of-function mutations around transmembrane regions of αIIb or β3 are associated with congenital macrothrombocytopenia, which is referred to as αIIbβ3-related thrombocytopenia. αIIb(R995W) is the most prevalent mutation in αIIbβ3-related thrombocytopenia among Japanese (Kunishima, Kashiwagi, et al. Blood. 2011;117:5479-84). We also found reduction of surface expression of αIIbβ3 on platelets in patients with αIIbβ3-rerated thrombocytopenia, suggesting that activating mutations may affect platelet function via not only activating state of αIIbβ3 but also its expression (Kashiwagi, et al. Mol Genet Genomic Med. 2013;1:77-86). However, the pathogenesis of thrombocytopenia caused by αIIbβ3 gain-of-function mutations is not fully understood. We have generated αIIb(R990W), which is corresponding to human αIIb(R995W), knock-in (KI) mice and previously reported that αIIb(R990W) KI mice showed mild macrothrombocytopenia and Homo KI mice revealed marked reduction of surface αIIbβ3 expression in platelet and impaired platelet function like GT (ASH meeting 2014). In this study, we further analyzed αIIb(R990W) KI mice to elucidate the mechanism causing macrothrombocytopenia.Methods:αIIb(R990W) mutation was introduced to C57BL/6-derived ES cells by homologous recombination and KI mice were generated. Reticulated platelets were detected by flow cytometry after thiazole orange staining. Serum thrombopoietin (TPO) levels were measured by ELISA. Platelet life span was assessed by flow cytometry after injection with NHS-biotin. Platelet counts and reticulated platelets were monitored after injection of murine TPO or anti-GPIb antibody. We counted megakaryocytes (MgK) in tissue preparation of femur and spleen. MgK ploidy was analyzed by flow cytometry. We analyzed proplatelet formation (PPF) of cultured fetal liver cells from embryonic day 14.5 embryos.Results:Platelet counts were decreased in KI mice [WT: 1.22 ± 0.11 (x106/μL), Hetero: 1.10 ± 0.14*, Homo: 0.91 ± 0.11* (*p<0.05 compared with WT)]. Absolute reticulated platelet numbers were significantly decreased in Homo mice compared with WT [WT: 102 ± 21.9 (x103/μL), Hetero: 84.0 ± 23.7, Homo: 66.6 ± 12.3* (*p<0.05 compared with WT)]. Although serum TPO level tended to be high in Homo mice [WT: 374 ± 155 (pg/mL), hetero: 380 ± 83, homo: 430 ±283], there were no statistically significant differences. Platelet increase after TPO injection was significantly delayed and peak platelet counts were lower in Homo mice compared to WT and Hetero mice. Recovery from anti-GPIb antibody induced thrombocytopenia was mildly impaired on 4 days after injection in KI mice. These results suggest that platelet production is impaired in KI mice. In addition, the half-life of platelets was slightly but significantly shortened in KI mice compared with WT [WT: 28.99 ± 1.76 (hours), Hetero: 24.90 ± 4.73, Homo: 19.54 ± 1.01* (*p<0.05 compared with WT)], which may also contribute to thrombocytopenia in KI mice. Between Homo mice and WT, there was no difference in the number of MgK in femur bone marrow [WT: 68.7 ± 5.04 (/10 visual fields), Homo: 75.7 ± 6.74] and spleen [WT: 24.7 ± 5.21 (/slide), Homo: 28.3 ± 5.55]. MgK ploidy in KI mice were similar to WT. However, PPF of MgK derived from fetal liver cells was significantly decreased in Homo mice [WT: 24.94 ± 3.11 (%), hetero: 22.21 ± 1.55, homo: 15.65 ± 1.49* (*p<0.05 compared with WT)].Conclusion:Our results suggest that the αIIbβ3 gain-of-function mutation, αIIb(R990W), leads to thrombocytopenia via mainly impaired thrombopoiesis. There was no difference in number and ploidy of MgK. However, Homo KI mice show marked reduction of PPF, indicating impaired proplatelet formation appears to be a main cause of impairment of thrombopoiesis by the mutation.Disclosures:No relevant conflicts of interest to declare. DisclosuresKanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Tomiyama:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Consultancy.

Antisense Oligonucleotide Targeting of Thrombopoietin Synthesis Reduces Platelet Count within the Hemostatic Range and Slows Progression of De Novo Mammary Carcinogenesis in the Mmtv-Pymt Mouse

Platelets have long been proposed to play a role in fostering neoplasias, ranging from tissue repair to cancer, but clinical evidence remains limited. Since available therapautics antagonizing hemostatic platelet functions lead to bleeding, the concept of targeting platelets in the context of cancer prevention or treatment has not been pursued in controlled clinical trials. To address this, we developed a novel antiplatelet strategy by partial inhibition of thrombopoiesis.We hypothesized that if platelets played a key role in fostering neoplastic malignancies, controlled platelet count reduction within a safe range would slow platelet-dependent cancer progression without adversely affecting hemostasis. Since the MPL ligand megakaryocyte growth and development factor (thrombopoietin, MGDF) is required for maintenance of normal platelet count, and the liver is responsible only for a portion of thrombopoietin synthesis, we tested this hypothesis by targeted knock-down of hepatic thrombopoietin synthesis using an antisense oligonucleotide (ASO) in mammary cancer-prone transgenic mice. We investigated whether partial reduction of thrombopoietin sythesis through ASO knock-down was hemostatically safe and impacted kinetics or penetrance of neoplastic progression in de novo mammary carcinogenesis in MMTV-PyMT mice.We designed and generated thrombopoietin ASO-s (TPO ASO) to achieve about 50% platelet count reduction in mice by inhibition of hepatic thrombopoietin gene expression. Then MMTV-PyMT transgenic mice were subcutaneously administered one of the effective TPO ASO-s, with treatment initiated at 40 days of age and before appearance of palpable (>2.0 mm) mammary tumors. We assessed the impact of saturating dose TPO ASO, including platelet count, plasma TPO level, and number of megakaryocytes in the femoral bone marrow. Mammary tumor growth, pulmonary metastases, and overall survival (fixed end-point study based on total tumor burden) of TPO ASO-treated mice were compared with untreated controls. At study endpoints, mammary carcinomas, platelet deposition, intra-tumoral vessel density, and Ki-67 positive cells were quantitatively evaluated.As anticipated from the drug screen, TPO ASO treatment at saturating effect reduced plasma thrombopoietin levels and blood platelet count by ~50% within 4 weeks, and the number of megakaryocytes in femoral bone marrow. TPO ASO also suppressed primary tumor growth (attached figure) and impaired development of pulmonary metastases (p<0.05), resulting in increased overall survival (p<0.001). Histological analysis of primary tumors revealed that TPO ASO significantly reduced mean platelet deposition in tumor vessels (from 54% to 26%; p<0.001), reduced vessel density (from 3.1% area to 1.5% area; p<0.05) and Ki-67 positive cells (from 62/field to 36/field; p<0.05) in primary tumors. Together, these results support the notion that strategies that safely reduce platelet presence or activity within developing mammary tumors, may limit neoplastic progression coincident with reduced angiogenesis.In summary, we found that TPO ASO reduces thrombopoietin levels and platelet count within the hemostatically safe range and significantly slows neoplastic progression of spontaneous mammary carcinomas in MMTV-PyMT mice. Overall, pharmacological knockdown of hepatic TPO synthesis is a new antiplatelet strategy that may be reasonably safe and effective in various platelet-driven disorders, including cancer. DisclosuresRevenko:Ionis Pharmaceuticals, Inc: Employment, Other: Intellectual property rights. Monia:Ionis Pharmaceuticals, Inc: Employment, Other: Intellectual property rights. Gruber:Aronora, Inc: Employment, Other: Intellectual property rights.

CCL5 Enhances Megakaryocyte Differentiation and Development

The importance of platelets in clot formation is well understood, but the role of platelets in the inflammatory response is becoming increasingly apparent. Platelet count increases during acute inflammatory stress; platelets also facilitate inflammation by releasing chemokines and cytokines from their granules. Our lab has previously shown that platelet derived chemokine ligand 5 (CCL5, RANTES), an inflammatory chemokine that is stored in platelets and released upon activation, acts to increase megakaryocyte maturation and proplatelet production in vitro. Furthermore, our lab found that CCL5 treated megakaryocytes have enhanced phosphorylation of proteins in the AKT pathway, such as BAD, which when phosphorylated induces pro-survival signaling. This indicates that CCL5-induced increase of platelet production is mediated by suppression of apoptosis, but the exact mechanism of CCL5 on megakaryocytes is not understood.To more completely asses the global impact of CCL5 on megakaryocytes, we performed proteomic analysis of CCL5-treated megakaryocytes and found that, as expected, CCL5 increases the expression of several pro-survival proteins in the AKT pathway. We have now confirmed increased phosphorylation of BAD by western blot in megakaryocytes treated with CCL5. In addition, we found that CCL5 treatment protects megakaryocytes from etoposide-induced apoptosis. Together, our data support the model of CCL5-induced inhibition of mitochondrial apoptosis via BAD phosphorylation. Interestingly, proteomic analysis also revealed an increase in proteins involved in quantity and differentiation of hematopoietic progenitor cells, as well as differentiation of megakaryocytes. To test whether CCL5 can also affect the differentiation of hematopoietic progenitor cells, we cultured mouse bone marrow with 100 ng/mL CCL5 and TPO and found a small but sustained increase in the number of megakaryocytes in vitro. To better understand the effect of CCL5 on skewing of hematopoietic cells in a physiological setting, we serially injected mice with 0.025 mg/kg or 0.05 mg/kg CCL5 for one week. Flow cytometry of bone marrow from these mice showed an increase in the number of Pre-MK E (Lin-/cKit+/Sca-1-/CD41-/FcgR-/CD105-/CD150+) and Pre-CFU E (Lin-/cKit+/Sca-1-/CD41-/FcgR-/CD105+/CD150+) populations and a decrease in the number of Pre GM (Lin-/cKit+/Sca-1-/CD41-/FcgR-/CD105-/CD150-) cells in CCL5 treated mice. Interestingly, the CCL5 treated mice did not have a significant inflammatory response, as was measured by complete blood count, indicating that these hematopoietic changes are independent from inflammation. These data suggest that CCL5 may act to skew hematopoiesis toward a megakaryocyte-erythroid lineage and away from a granulocyte lineage in non-inflamed mice.Our proteomics data and in vitro live-dead assay suggest that CCL5 treatment protects against apoptosis through increased BAD phosphorylation, potentially leading to increased megakaryocyte maturation and proplatelet formation. Furthermore, CCL5 may have a dual action in both enhancing platelet production via pro-survival signaling and skewing hematopoiesis toward the megakaryocyte-erythroid lineage. Hematopoietic skewing was seen in CCL5 treated mice despite not having increased inflammation, suggesting that CCL5 itself does not cause a significant inflammatory response. Nonetheless, CCL5 has previously been shown to increase in an inflammatory setting, indicating that the effects seen here could be triggered by systemic inflammation in a different model. Further investigation of the mechanisms leading to these phenotypes may reveal novel therapeutic options to enhance megakaryocyte development and increase platelet counts in patients with thrombocytopenia. DisclosuresBattinelli:Sanofi: Consultancy.

Development of Liver-Specific Thrombopoietin Targeted Sirnas: Impact on Platelet Count, Megakaryocyte Mass, and Hematopoietic Progenitors in Normal and MPN Murine Models

The Thrombopoietin (THPO)/ thrombopoietin receptor (MPL) signaling axis is not only critical for the generation of platelets and megakaryocytes, but also for the maintenance of hematopoietic stem cells (HSC) and the bone marrow niche. MPL is expressed on primitive HSC, HSC progenitors, megakaryocytes, platelets, osteoblasts and osteoclasts, clonal hematopoietic stem cells and many leukemias. THPO production is constitutive but is also increased by inflammatory cytokines. Sustained exposure to high levels of THPO not only enhances platelet production, but also has a profound effect on HSC and the bone marrow microenvironment. Excess THPO/MPL signaling, whether driven by inflammatory cytokines, or due to mutations in THPO, JAK2, MPL or CALR, is associated with HSC expansion, megakaryocyte hypertrophy and increased platelet count, excess release of megakaryocyte and platelet-based cytokines such as TGF-beta and PDGF-alpha, and the development of stromal myofibroblasts that drive tissue fibrosis and anemia.We developed a robust and clinically validated RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent N-acetylgalactosamine (GalNAc) conjugates, enabling specific silencing of hepatocyte-expressed genes following subcutaneous injection. Since liver is the major source of THPO expression, we utilized GalNAc-siRNA technology to develop siRNAs targeting THPO for evaluation in wild type mice and murine models of myeloproliferative neoplasms (MPN). Active siRNAs were identified by in vitro screening in primary mouse hepatocytes and the 12 best siRNAs were evaluated in vivo in normal mice to select the most potent siRNA. THPO liver mRNA levels were reduced by up to 80% after a single subcutaneous THPO siRNA dose, with no effect on THPO mRNA expression in other organs (kidney, and bone marrow, both of which had marginal THPO expression compared to liver). Circulating TPO levels were reduced by 80% by day 7 and were suppressed for up to 28 days post a single dose treatment. Platelet counts were reduced to 60% of baseline by day 14, and a further reduction to more than 70% of baseline was observed with every other week dosing. No changes in red blood cell or white blood cell subsets were observed. Platelet reduction was accompanied by a reduction in megakaryocyte mass, as evidenced by a 50% decrease in the number of bone marrow megakaryocytes in THPO siRNA treated mice compared to controls. Mice treated every other week with TPO siRNA for three months demonstrated sustained circulating THPO protein and platelet count reductions, and a significant reduction in bone marrow HSC, Lin-Sca1+Kit- (LSK) and multipotent progenitor (MPP) frequency. Evaluation of impact THPO silencing on MPN phenotypes in transgenic JAK2V617F mice is ongoing. THPO silencing is a potential novel targeted therapeutic approach that may be beneficial in benign and malignant conditions in which deregulated THPO/MPL signal transduction drives disease pathology. DisclosuresNo relevant conflicts of interest to declare.

Hippo Kinase Inactivation Contributes to Del(20q) Malignancies and Cooperates with JAK2-V617F to Promote Myelofibrosis in Mice

Recurring chromosome abnormalities are frequent events in cancer and are especially prevalent in hematologic neoplasms. Somatic heterozygous deletions on chromosome 20q are detected in a variety of hematopoietic malignancies including myelodysplastic syndrome (MDS), classical myeloproliferative neoplasm (MPN), MDS/MPN overlap disorders such as chronic myelomonocytic leukemia (CMML), and acute leukemias. Del(20q) is especially prevalent in MPN patients (~10-15%), where it is the most commonly detected cytogenetic abnormality associated with primary myelofibrosis (PMF) and post-polycythemia vera myelofibrosis (MF). This suggests that heterozygous loss of genes in the del(20q) common deleted region (CDR) may contribute to adverse MPN progression. Despite these observations, relatively few genes located within the CDR have been unambiguously implicated, highlighting a significant need for further investigation. To identify genes that may play an important role in the biology of del(20q)-associated malignancies we utilized a published gene expression dataset of bone-marrow derived CD34+ cells from MDS patients and healthy controls (Gerstung et al, 2015). Comparison of the patients harboring del(20q) to healthy controls revealed STK4 (encoding Hippo kinase MST1) to be the most significantly downregulated gene (mean: 3.5-fold) among those located within the chromosome 20q CDR. We therefore set out to assess the role of Hippo kinase inactivation in hematologic malignancy using conditional gene inactivation in mice.We found that complete inactivation of both Hippo kinases (Stk4 and Stk3) within the hematopoietic system using Vav1-Cre (Stk4-/-Stk3-/-) resulted in a lethal bone marrow failure (median survival: 7 weeks) associated with myelodysplastic features and frequent extramedullary hematopoiesis in the spleen. A single copy of Stk4 rescued the lethality due to bone marrow failure, however sub-haploinsufficient mice displayed thrombocytopenia with a trend towards mild anemia; phenotypes that closely resemble those observed in MDS patients with isolated del(20q). Both a reduced number of mature megakaryocytes and the presence of dysplastic megakaryocytes were apparent in bone marrow sections. Inducible Hippo kinase inactivation in adult mice using the Mx1-Cre system similarly recapitulated several phenotypic features of both MDS and MPN. In competitive bone marrow transplant assays we found that Stk4-/-Stk3-/- hematopoietic stem cells (HSC) completely lacked engraftment potential and failed to reconstitute normal hematopoiesis, revealing a potential role for Hippo kinase function in HSC homing and retention in the bone marrow. Heterozygous HSCs maintained relatively normal steady-state hematopoiesis in peripheral blood and bone marrow for up to 48 weeks in primary and secondary transplantations, although upon aging these mice were prone to development of thrombocytopenia with increased mean platelet volume.Given the high frequency of del(20q) in MPN, especially PMF, we asked whether heterozygous Hippo kinase inactivation may cooperate with the common driver mutation JAK2-V617F to accelerate disease progression. Using an HSC-enriched retroviral transduction/transplantation model in C57BL/6 recipient mice, we monitored MPN progression for 36 weeks in heterozygous Stk4+/-Stk3+/-, or control (Vav1-Cre-), cells with or without expression of JAK2-V617F. While both JAK2-V617F groups initially displayed a similar degree of polycythemia relative to controls, we found heterozygous Hippo kinase inactivation to promote accelerated disease progression towards lethal bone marrow fibrosis during the course of observation. Recipients in this group showed significantly reduced overall survival, which was associated with higher grade fibrosis in bone marrow, elevated peripheral granulocyte counts, enhanced splenomegaly, and increased frequencies of hematopoietic stem and progenitor populations in the spleen. Together, these findings implicate aberrant Hippo kinase loss-of-function in the pathogenesis of del(20q)-associated hematologic malignancies, and shed new light on the molecular events that contribute to adverse MPN progression. DisclosuresBejar:Genoptix: Consultancy; Modus Outcomes: Consultancy; Celgene: Consultancy, Honoraria; Takeda: Research Funding; Astex/Otsuka: Consultancy, Honoraria; AbbVie/Genentech: Consultancy, Honoraria; Foundation Medicine: Consultancy. Guan:Vivace: Equity Ownership.

Alisertib (MLN8237), an Oral Selective Inhibitor of Aurora Kinase a, Has Clinical Activity and Restores GATA1 Expression in Patients with Myelofibrosis

Alisertib (MLN8237), an Oral Selective Inhibitor of Aurora Kinase a, Has Clinical Activity and Restores GATA1 Expression in Patients with Myelofibrosis

Distinct Microrna Profiles in Patients with Essential Thrombocythemia: Clinical and Prognostic Correlation

Background: Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm characterized by increased number of mature megakaryocytes in the bone marrow and sustained thrombocytosis in the peripheral blood. ET is associated with an increased risk of hemorrhagic and thrombotic complications and leukemic transformation. In addition to somatic mutations, microRNA (miRNA) deregulation has been proposed to play roles in the molecular pathogenesis and disease phenotype in ET patients. The aims of this study were to investigate the plasma miRNA profiles in ET patients with those obtained from healthy adults (HA) and its correlation with clinical and prognostic features.Methods: ET patients seen at the MacKay Memorial Hospital were enrolled into this study. The clinical and laboratory characteristics at the time of diagnosis or referral were determined retrospectively by chart review. Total plasma miRNA was derived from bone marrow or peripheral blood in patients or HA. Mutational status of JAK2V617F,CALR and MPL were detected by Sanger sequencing and/or allele-specific PCR. Plasma miRNA profiling was carried out on PanelChip™ Analysis System (Quark Biosciences, Taiwan) using a customized miRNA panel including 165 miRNAs called mirSCAN™ PanCancer Chips 1 & 2. KEGG pathways that miRNAs were associated with were analyzed using DIANA TOOLS - mirPath v.3. Differentially expressed miRNAs were identified by student t-test (P < 0.05) and significantly affected miRNAs in ET were identified by a change in expression of two-fold or more compared to the expression in HA. Statistical analysis was performed using SPSS.Results: A total of 53 ET patients (median age at diagnosis 59 years; 60.4% females) were enrolled. Frequency of the 3 driver mutations was 64% for JAK2V617F, 15% CALR, 4% JAK2V617F and CALR co-mutations, and none for MPL. 17% of patients were classified as triple-negative. A total of 40 differentially expressed miRNAs were identified (Figure left axis) including 4 miRNAs (hsa-miR-940, hsa-miR-411-5p, hsa-miR-596 and hsa-miR-376c-3p) with higher expression levels. The putative target genes of these 40 differentially expressed miRNAs were enriched in signaling pathway including TGF-beta signaling pathway (p-value = 1.86E-12), Hippo signaling pathway (p-value = 1.84E-07), MAPK signaling pathway (p-value = 0.03), and FoxO signaling pathway (p-value < 0.001). According to the expression levels of these 40 miRNAs, samples were grouped into two clusters, i.e. low expression group and high expression group. ET patients with JAK2V617F were significantly associated with high expression of 40 miRNAs (n = 31 of 34, 91%) when compared with triple-negative ET patients (n = 5 of 9, 56%) (p = 0.03). ET patients with low expression of 40 miRNAs were significantly associated with acute leukemia transformation (n = 2 of 11, 18%) when compared with high expression group (n = 0 of 42, 0%) (p = 0.04). However, the expression levels of 40 miRNAs were not statistically correlated with other clinical features including hemogram, secondary solid cancer, myelofibrosis transformation, or thrombotic/hemorrhagic events.Conclusions: In this cohort of ET patients, distinct plasma miRNA profiles correlated with JAK2V617F mutation and leukemic transformation. Larger cohort is warranted to validate our findings. [Display omitted] DisclosuresChen:Quark Biosciences, Inc.: Employment. Kang:Quark Biosciences, Inc.: Employment. Huang:Quark Biosciences, Inc.: Employment. Lin:Quark Biosciences, Inc.: Employment. Wang:Quark Biosciences, Inc.: Employment.

Genotype-Resultant Morphology of Myelodysplastic Syndromes (MDS)

Morphology has dominated the diagnosis and classification of MDS for decades. With the advent of NGS, morphology is used as a gold standard to assess associations of specific disease sub-entities with individual mutations or their combinations. An example is the association between SF3B1 mutations and ringed sideroblasts, or with disease-defining lesions, such as t(8:21), 11q23 translocations, and EVI1 mutations. Beyond these discrete examples, assignments of diverse morphologic features to combinations of specific genotypes has been more challenging.To identify potentially novel paradigms of associations between molecular hits and morphologies we examined 835 patients with MDS. Common somatic mutations, chromosomal abnormalities and morphologic reviews were subjected to blinded, objective standard diagnostic criteria. Abnormalities included individual lineage dysplasia and cytopenias, monocytosis, fibrosis, and increased megakaryocytes, all classified using standard criteria. To correlate the presence of mutations to morphologic criteria, only mutations with a clonal burden >10% were used. In total, 794 out of 835 patients (95%) harbored at least one lineage dysplastic feature. Myeloid, erythroid and megakaryocytic dysplasia occurred in 56% (470/835), 71% (590/835) and, 70% (587/835) of patients, respectively. Bi- and tri-lineage dysplasia were also identified in 36% (297/835) and 33% (278/835). In 90% (753/835) of patients there was cytopenia: 48% (402/835) had neutropenia, 61% (510/835) anemia, and 61% (510/835) thrombocytopenia. More than 50% of patients had multiple-cytopenias; 23% (194/835) had pancytopenia. Monocytosis was present in 20% (168/835) of patients and 31% (256/835) had elevated numbers of megakaryocytes. For analysis all possible combinations (25 pairs in total) were queried and those present in >80 patients were evaluated. Based on NGS among 36 genes, TET2 (27%), SF3B1 (23%), ASXL1 (19%), del(5q) (16%), SRSF2 (14%), DNMT3A (11%) and del(7q) (10%), were frequently mutated genes/CNAs, each present in >10% of patients. A number of relationships between phenotypic features and molecular context were uncovered. For example, patients with thrombocytopenia also commonly had tri-lineage dysplasia [38% (198/510) vs. 25%, (80/325), p<.001, q<.001] or more myeloid dysplasia [63% (321/510) vs. 46% (149/325), p<.0001, q<.0001] but no megakaryocyte dysplasia [72% (366/510) vs. 68% (221/325), p=.027]. Moreover lineage specific dysplasia was not associated with lineage specific cytopenias. In fact, myeloid or erythroid dysplasia had no significant association with neutropenia or anemia (p=.14 or p=.1, respectively).When we performed statistical analyses that sought correlations between clinical features and genetic alterations, 78 significant associations were identified. For instance, patients with erythroid dysplasia more frequently had SF3B1 mutations [in 15% of erythroid dysplasia patients, 90/590] than patients without it [6%, 15/245] (p=.0002, q=.001). In contrast, these patients were less likely to have EZH2 mutations [4.2% (25/590) vs. 11.4% (28/245), p<.001, q=.002]. Compared to patients with no myeloid dysplasia, patients with myeloid dysplasia were more likely to have TET2, STAG2, and SRSF2 mutations, and less likely to have SF3B1 mutations (p<.02). Compared to patients without megakaryocytic dysplasia, those with it more commonly had ASXL1, mutation, but less commonly SF3B1 and BCORL1 mutations. Patients with STAG2 mutations [8%, 66/835] were more likely to have neutropenia [62% (41/66) vs. 47% (361/769), p=.02, q=.08]. TP53 and ASXL1 mutations occurred frequently in patients with thrombocytopenia. Monocytosis co-existed more with 5 genetic mutations, including SRSF2 and RAS pathway genes.Using various conventional and more advanced strategies, we present the first comprehensive genotype-phenotype association study that linked somatic molecular events to morphologic and clinical features. We demonstrate that the complexities of how molecular context governs morphologic changes can be deciphered. Certain rigorously defined morphologic configurations show specific associations with individual mutations or their combinations. DisclosuresNazha:MEI: Consultancy. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

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.

Eltrombopag Promotes Megakaryocyte Survival and Signaling in the Presence of Specific Cytotoxic Agents

BACKGROUND: Cytotoxic chemotherapy/agents can cause a range of side effects in cancer patients including anemia, neutropenia and thrombocytopenia, resulting in increased morbidity and mortality. These cytopenias are in part due to massive depletion of bone marrow progenitors. A common target of many chemotherapies is the megakaryocyte (MK), a rare progenitor representing 0.2% of the bone marrow. Loss of MK lineage cells results in thrombocytopenia, therefore, discovering ways to protect MKs from cytotoxic drugs could prevent this life-threatening condition. Here, we show that eltrombopag (EP), a small molecule, non-peptide thrombopoietin receptor (TPO-R) agonist, protects healthy MKs from different cytotoxic agents. We further explored the impact of cytotoxic drugs and EP treatment on signaling molecules active in MKs.METHODS: Peripheral blood and bone marrow samples were collected from healthy donors after informed consent and following protocols in accordance with the Declaration of Helsinki. CD34+ positive cells were isolated by immuno-magnetic bead separation from the samples and expanded for 8-10 days with a cytokine cocktail to induce MK differentiation. After MK expansion, cells were transferred to 96-well plates pretreated with 14 different drugs including cytarabine, gemcitabine, paclitaxel, carboplatin, cisplatin, doxorubicin, vincristine, etoposide, midostaurin, ruxolitinib, panobinostat, azacitidine, venetoclax and navitoclax. The drugs were plated in 5 different doses in a 10,000-fold concentration range, and cells added with or without EP. After 3 days incubation, the cells were analyzed on a high throughput flow cytometer using Annexin V and 7AAD to distinguish live from dead cells. To understand the impact of the drugs and EP on signaling molecules downstream of TPO-R, we analyzed phosphorylation of AKT, ERK, STAT3 and STAT5 in populations defined by MK markers CD41a, CD42b and CD110 (TPO-R).RESULTS: We found that EP overall supports MK survival in the presence of cytotoxic agents. The greatest net effect was observed when EP was combined with BCL2 inhibitors (venetoclax, navitoclax), which resulted in increased MK cell maturation compared to inhibitor alone, while gemcitabine plus EP showed the next best response. EP combined with etoposide, vincristine, cytarabine, paclitaxel, cisplatin and ruxolitinib resulted in an increase of immature MKs without a reduction in mature MK cell numbers. Higher numbers of MKs were observed when EP was combined with midostaurin, carboplatin, panobinostat and doxorubicin, compared to treatment without EP; although these numbers were lower than other tested drugs. Phosphoflow analysis revealed intriguing differences in EP-induced signaling compared to signaling induced by recombinant human thrombopoietin (rhTPO) (Figure 1). Also, EP-induced signaling did not correlate with TPO-R expression. EP activated AKT in all MK subsets, but rhTPO induced AKT only in immature MKs. While rhTPO induced ERK phosphorylation in different MK subsets, EP had no effect on ERK activation. Furthermore, rhTPO activated STAT3 only in mature MKs, but EP induced STAT3 in all MK populations. In contrast, both EP and rhTPO induced phosphorylation of STAT5 in all MK subsets. Overall signaling was inhibited by a selected set of agents (venetoclax, etoposide, midostaurin) compared to basal signaling, but addition of EP prevented the inhibition. Similar to the cell survival analysis, venetoclax combined with EP resulted in complete recovery of signaling activity compared to venetoclax alone. But the response was not as striking for etoposide or midostaurin combination compared to treatment alone.CONCLUSION: Using MKs expanded from CD34+ cells, we found that EP supports MK survival in the presence of several different cytotoxic agents, with the most striking rescue observed when EP was combined with BCL2 inhibitors. Importantly, EP and rhTPO induced distinct signaling patterns, with EP signaling independent of TPO-R expression, suggesting alternate EP targets. The tested drugs inhibited basal signaling in MKs, but this inhibition was prevented by EP. These results suggest addition of EP to cancer treatment regimens may prevent the inhibitory effects of some agents on MKs and support future investigations to demonstrate clinical efficacy of EP in combinations with some of these cytotoxic agents to alleviate thrombocytopenia. [Display omitted] DisclosuresJavarappa:Novartis Pharma AG: Research Funding. Tsallos:Novartis: Research Funding. Marques Ramos:Novartis: Employment. Pallaud:Novartis: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Orion Pharma: Research Funding.

Spatial Regulation of Thrombopoiesis in the Bone Marrow

Blood platelets play key roles in hemostasis and thrombosis and are the second most abundant cell type in the circulation. Due to their short life span of only a few days, anuclear platelets are continuously replenished and thus provide a classic system to study hematopoiesis. In mammals, platelets are produced by megakaryocytes (MKs) that are predominantly residing in the bone marrow (BM). MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Unfortunately, previous studies on megakaryopoiesis were often limited by 2D imaging and cutting artefacts when analyzing bone sections, potentially resulting in underestimation of MK-to-vessel contacts and MK volumes. We studied megakaryopoiesis by visualizing MKs in their 3D environment. To this end, murine bones were simultaneously stained for MKs and endothelial cells, fixed, chemically cleared and imaged by Light Sheet Fluorescence Microscopy (LSFM). Thus, we achieved 3D-reconstructions of the complete and intact bone with subcellular resolution. Through imaging of MKs in the intact BM, we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. We developed and compared different image processing pipelines and simulation scenarios for precise identification of MKs in 3D light-sheet fluorescence microscopy of uncut murine bones. By combining in vivo two-photon microscopy and in situ LSFM with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. To complement limited imaging approaches computational simulations represent an important, well-controllable tool. Typically, simulation studies use artificial meshes as templates to minimize the computational effort or due to the lack of experimental data. Unfortunately, such simplified artificial templates for MKs and the vasculature can bias simulations and lead to misinterpretations as we show here. Using the segmented cell and vessel objects of true 3D images can overcome those limitations providing a simulation framework that has the prerequisites to maximally reflect the physiological situation. Thus, imaging and simulations go hand in hand when the respective 3D cell and vessel objects perfectly serve as biological templates for advanced simulations. We demonstrate reliable whole-bone analysis in silico, and found that MKs influence neutrophil and HSC migration as biomechanical restrainers modulating cell mobility and extravasation. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche (1). Furthermore, we identify MKs as biomechanical restraints for bone marrow cell mobilization. As MKs themselves do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.(1) Stegner D, van Eeuwijk JMM, Angay O, Gorelashvili MG, Semeniak D, Pinnecker J, Schmithausen P, Meyer I, Friedrich M, Dütting S, Brede C, Beilhack A, Schulze H, Nieswandt B, Heinze KG. Thrombopoiesis is spatially regulated by the bone marrow vasculature, Nat Commun. 2017 8(1):127. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of profibrotic microRNA-21 affects platelets and their releasate

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21–null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21–null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.