Long-acting PGE2 and Lisinopril Mitigate H-ARS.

Thrombocytopenia is a major complication in hematopoietic-acute radiation syndrome (H-ARS) that increases the risk of mortality from uncontrolled hemorrhage. There is a great demand for new therapies to improve survival and mitigate bleeding in H-ARS. Thrombopoiesis requires interactions between megakaryocytes (MKs) and endothelial cells. 16, 16-dimethyl prostaglandin E2 (dmPGE2), a longer-acting analogue of PGE2, promotes hematopoietic recovery after total-body irradiation (TBI), and various angiotensin-converting enzyme (ACE) inhibitors mitigate endothelial injury after radiation exposure. Here, we tested a combination therapy of dmPGE2 and lisinopril to mitigate thrombocytopenia in murine models of H-ARS following TBI. After 7.75 Gy TBI, dmPGE2 and lisinopril each increased survival relative to vehicle controls. Importantly, combined dmPGE2 and lisinopril therapy enhanced survival greater than either individual agent. Studies performed after 4 Gy TBI revealed reduced numbers of marrow MKs and circulating platelets. In addition, sublethal TBI induced abnormalities both in MK maturation and in in vitro and in vivo platelet function. dmPGE2, alone and in combination with lisinopril, improved recovery of marrow MKs and peripheral platelets. Finally, sublethal TBI transiently reduced the number of marrow Lin-CD45-CD31+Sca-1- sinusoidal endothelial cells, while combined dmPGE2 and lisinopril treatment, but not single-agent treatment, accelerated their recovery. Taken together, these data support the concept that combined dmPGE2 and lisinopril therapy improves thrombocytopenia and survival by promoting recovery of the MK lineage, as well as the MK niche, in the setting of H-ARS.

In vitro large scale production of megakaryocytes to functional platelets from human hematopoietic stem cells.

Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32 N while TPO induced MKs showed polyploidy of 8 N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.

Justicia adhatoda induces megakaryocyte differentiation through mitochondrial ROS generation.

BACKGROUND: Hepatoprotective activity along with improved survival percentage and hematological parameters prior to whole body irradiation were reported with Justicia adhatoda extracts. PURPOSE: To evaluate the thrombopoietic potential of Justicia adhatoda L. leaf extract in megakaryocyte differentiation METHODS: Ethanol extracts were prepared using soxhlet extraction method, and IC50 value was determined. The effect of ethanol extracts obtained from Justicia adhatoda on megakaryocyte maturation and development in megakaryocytic Dami cell lines was tested. Expression of megakaryocyte specific markers, CD61 and CD41, were assessed using flow cytometry and fluorescence microscopy. In addition, cell cycle analysis and mitochondrial membrane potential were analyzed by flow cytometry. Gene expression analysis was performed using qRT-PCR. RESULTS: At a concentration of 40 µg/ml, the leaf extracts of Justicia adhatoda for 72 h induced the megakaryocytic features in megakaryocytic Dami cell lines. The megakaryocyte specific markers, CD41 and CD61, were up-regulated (2.2 and 12.4 fold, respectively), and more number of cells entered into synthetic (S) and G2/M phase as compared with untreated cell (23.1% vs 16.6% and 70.2% vs 42.3%, respectively) showing maturation. RUNX1 (a transcription factor essential for embryonic hematopoiesis and adult megkaryocyte maturation) and c-Mpl (the receptor for TPO) were upregulated, and the suppressor of cytokine signaling (SOCS) 1 and SOCS3 were down-regulated upon treatment with Justicia adhatoda. Justicia adhatoda enhanced mitochondrial ROS generation by 28-fold, increased the permeability of mitochondrial membrane and showed an inverse correlation in superoxide dismutase levels. CONCLUSION: Justicia adhatoda could enhance mitochondrial ROS generation and increase the permeability of mitochondrial membrane, thereby inducing megakaryocytic maturation. Our findings suggest thrombopoietic potential of Justicia adhatoda leaf extract on megakaryocyte differentiation.

Ocimum flavone Orientin as a countermeasure for thrombocytopenia.

Thrombocytopenia or chronic depletion of platelets in blood, could create life-threatening conditions in patients who receive aggressive systemic radiation and chemotherapy. Currently there are no approved agents for the rapid treatment of thrombocytopenia. In the present study, we demonstrate that administration of Orientin, a glycosidic flavonoid or dietary administration of Orientin containing Tulsi (Holy Basil) leaves, results in a significant increase in circulating platelets in a clinically relevant mouse model. No noticeable effects were observed on red blood cells, white blood cells or other hematologic parameters in treated animals indicating that Orientin specificity enhances platelet formation. The gene expression and immunophenotyping of bone marrow revealed that Orientin stimulates megakaryopoiesis specific transcriptional program. A significant increase in colony formation in bone marrow cells from Orientin pretreated mice further complemented the effect of Orientin on progenitor cells. The ex-vivo differentiation of irradiated human peripheral blood CD34+ stem cells demonstrated stimulatory effects of Orientin on megakaryocyte erythrocyte progenitors (MEP). The results show that Orientin, a non-toxic readily available natural product can counter platelet imbalances. Thrombocytopenia also develop as a consequence of multiple hematologic malignancies and side effects of treatments. Dietary supplementation of Orientin containing phytochemicals could be effective as countermeasures and viable therapeutics.

A novel mode of stimulating platelet formation activity in megakaryocytes with peanut skin extract.

We report in this study novel biochemical activities of peanut skin extract (PEXT) on thrombocytopoiesis. Peanut skin, derived from Arachis hypogaea L., is a traditional Chinese medicine that is used to treat chronic hemorrhage. We have shown that oral administration of PEXT increases the peripheral platelet levels in mice. Recently, we reported a liquid culture system that is useful for investigating megakaryocytopoiesis and thrombocytopoiesis from human CD34+ cells. In this liquid culture system, PEXT was shown to enhance the formation of CD41+/DAPI- cells (platelets), but had no effect on the formation of CD41+/DAPI+ cells (megakaryocytes) or on the DNA content. Furthermore, PEXT selectively stimulated proplatelet formation from cultured mature megakaryocytes and phorbol 12-myristate 13 acetate (PMA)-induced formation of platelet-like particles from Meg01 cells. Despite having no influence on the formation of megakaryocyte colony forming units (CFUs), PEXT increased the size of megakaryocytes during their development from CD34+ cells. PEXT showed no effect on the GATA-1 and NF-E2 mRNA levels, which are known to play an important role in thrombocytopoiesis and, based on the results of a pMARE-Luc (pGL3-MARE-luciferase) assay, had no influence on NF-E2 activation in Meg01 cells. These results suggest that PEXT accelerates proplatelet formation from megakaryocytes but does not influence the development of hematopoietic stem cells into megakaryocytes.

Preliminary nonclinical toxicity, pharmacokinetics, and pharmacodynamics of ALXN4100TPO, a thrombopoietin receptor agonist, in CD2F1 mice.

ALXN4100TPO, a thrombopoietin (TPO) receptor agonist, increases platelets, abrogates radiation-induced thrombocytopenia and affords significant survival benefit to lethally irradiated mice. This preliminary nonclinical safety study assessed effects of a single subcutaneous (sc) administration of ALXN4100TPO in CD2F1 mice randomized into naïve, control antibody (ALXN4200, 100 mg/kg), low (1 mg/kg), medium (10 mg/kg), or high (100 mg/kg) ALXN4100TPO doses. End points included clinical observations, body weight changes, hematology, histopathology, pharmacokinetics, pharmacodynamics by measuring platelet counts, and endogenous TPO (eTPO) levels. Salient findings were prominent increase in platelet counts and end cells of myeloid and lymphoid lineages; elevated megakaryopoiesis in bone marrow; and extramedullary hematopoiesis in spleen and liver. Serum ALXN4100TPO levels were maximum 24 hours after administration, with a half-life of 13 days. Endogenous TPO levels were elevated in 10 and 100 mg/kg ALXN4100TPO-treated groups. In conclusion, ALXN4100TPO (1-100 mg/kg, sc) treatment in CD2F1 mice resulted in profound pharmacological changes in the hematopoietic tissue; however, no life-threatening adverse events were observed.

Polysaccharides from the root of Angelica sinensis promotes hematopoiesis and thrombopoiesis through the PI3K/AKT pathway.

BACKGROUND: Dozens of Traditional Chinese Medicine (TCM) formulas have been used for promotion of "blood production" for centuries, and we are interested in developing novel thrombopoietic medicines from these TCMs. Our previous studies have demonstrated the hematopoietic effects of DangGui BuXue Tong (DBT), a formula composed of Radix Angelicae Sinensis and Radix Astragali in animal and cellular models. As a step further to identify and characterize the active chemical components of DBT, we tested the hematopoietic and particularly, thrombopoietic effects of polysaccharide-enriched fractions from the root of Radix Angelicae Sinensis (APS) in this study. METHODS: A myelosuppression mouse model was treated with APS (10 mg/kg/day). Peripheral blood cells from APS, thrombopoietin and vehicle-treated samples were then counted at different time-points. Using the colony-forming unit (CFU) assays, we determined the effects of APS on the proliferation and differentiation of hematopoietic stem/progenitor cells and megakaryocytic lineages. Using a megakaryocytic cell line M-07e as model, we analyzed the cellular apoptosis progression with and without APS treatment by Annexin V, Mitochondrial Membrane Potential and Caspase 3 assays. Last, the anti-apoptotic effect of APS on cells treated with Ly294002, a Phosphatidylinositol 3-Kinse inhibitor (PI3K) was also tested. RESULTS: In animal models, APS significantly enhanced not only the recovery of platelets, other blood cells and their progenitor cells, but also the formation of Colony Forming Unit (CFU). In M-07e cells, we observed the anti-apoptotic effect of APS. Treatment by Ly294002 alone increased the percentage of cells undergoing apoptosis. However, addition of APS to Ly294002-treated cells significantly reduced the percentage of cells undergoing apoptosis. CONCLUSIONS: APS promotes hematopoiesis and thrombopoiesis in the mouse model. This effect likely resulted from the anti-apoptosis activity of APS and is likely to involve the PI3K/AKT pathway.

Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL.

OBJECTIVE: The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. MATERIALS AND METHODS: hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. RESULTS: During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. CONCLUSION: This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.

An herbal decoction of Radix astragali and Radix angelicae sinensis promotes hematopoiesis and thrombopoiesis.

ETHNOPHARMACOLOGICAL RELEVANCE: A decoction containing Radix angelicae sinensis and Radix astragali (Danggui Buxue Tang, DBT) has been used to raise the "Qi" and nourish the "Blood". However, its effects on haematopoiesis and particularly thrombopoiesis have not been studied. AIMS: This study aims to examine the effects of DBT on hematopoiesis and thrombopoiesis. MATERIALS AND METHODS: A myelosuppression mouse model was treated with DBT (10mg/kg/day). Peripheral blood cells from DBT and thrombopoietin-treated samples were counted on days 0, 7, 14 and 21. Then CFU assays were used to determine the effects of DBT on the megakaryocytic progenitor cells and other lineages. Last, analyses of annexin V, caspase-3, and mitochondrial membrane potential were conducted in megakaryocytic cell line M-07e. RESULTS: Morphological examination showed that DBT treatment significantly increased the recovery of the megakaryocytic series. DBT significantly enhanced the platelet recovery and CFU-MK formation in vivo. DBT significantly promoted CFU-MK and CFU-F formation. Last, we observed the antiapoptotic effects of DBT on M-07e cells. CONCLUSION: DBT might promote haematopoiesis and thrombopoiesis in the mouse model through (i) directly promoting the growth of megakaryocytes; (ii) indirectly promoting the growth of bone marrow stromal cells; (iii) inhibiting apoptosis of megakaryocytes.