561 Renin-angiotensin system expression in myeloproliferative diseases

Myeloproliferative diseases present the group of clonal malignant diseases of hematopoietic stem cell. Somatic mutation of JAK2 gene (JAK2V617F+) is present in most of the patients (>90%) with polycythemia vera (PV), and 50% of patients with essential thrombocythemia (ET). This mutation causes the constitutive activation of tyrosine kinase and the consequence is cytokine independent proliferation of cells. Signaling pathway JAK2/STAT5/BclxL is essential for erythropoiesis, controlling cell proliferation and survival. In JAK2V617F+ PV and ET, growth of erythroid progenitors is erythropoietin independent. There is a lot of evidence of local renin-angiotensin system (RAS) presence in bone marrow affecting cell proliferation and differentiation. There is an increase of mRNA expression of angiotensinogen (AGT), rennin (REN) and angiotensin II receptor 1 (AT2R1) in bone marrow of JAK2V617F+ PV and ET patients. Our research is focused on understanding the correlation of these two pathways in order to find the control points that can be used as a drug targets for myeloproliferative disorders. Bone marrow mononuclear cells from PV and ET patients were seeded on MethoCult (StemCell) in medium with and without erythropoietin (EPO). At day 13 erythroid colonies were observed for morphology differences, colony density and isolation of DNA, RNA and proteins (Trizol, Invitrogene). DNA is used to determine JAK2 status by allele-specific PCR, RNA for real-time PCR detection of RAS components and proteins for Western detection of AT2R1 protein. We analyzed 5 different bone marrow samples by now – 3 PV (JAK2V617F+), 1 PV (JAK2V617F−) and 1 ET (JAK2V617F+). JAK2V617F+ PV and ET erythroid colonies grown without EPO were smaller (50–100 cells), paler and in lower density then erythroid colonies grown with EPO (>200 cells). No erythroid colonies were noticed in JAK2V617F− PV sample without EPO. We collected high quality DNA, RNA and protein from cca 104 cells. We modified protein extraction method to gain better solubilization of proteins by resuspending them in 2% DEA. Preliminary results suggest increase of AT2R1 expression in JAK2V617F+ patients when compared with JAK2V617F− patients. Western and RT-PCR data are in preparation. Confirimation of higher expression of RAS components in patients that have constitutive activation of JAK2 will enable us to use drugs like ACE inhibitors and AT2R1 antagonists in assessing erythroid proliferation-differentiation process.