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. Disclosures No relevant conflicts of interest to declare.

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

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.

Aggressive Systemic Mastocytosis in Association with Pure Red Cell Aplasia

Aggressive systemic mastocytosis (ASM) is characterized by mast cell accumulation in systemic organs. Though ASM may be associated with other hematological disorders, the association with pure red cell aplasia (PRCA) is rare and has not been reported. Pure red cell aplasia (PRCA) is a syndrome, characterized by normochromic normocytic anemia, reticulocytopenia, and severe erythroid hypoplasia. The myeloid and megakaryocytic cell lines usually remain normal. Here, we report an unusual case of ASM, presenting in association with PRCA and the management challenges.

Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms

Key Points Mutant CALR induces TPO-independent growth in the human megakaryocytic cell line UT-7/TPO. Mutant CALR binds to the TPO receptor, inducing phosphorylation of JAK2 and activating downstream signaling.