Bojana B. Beleslin-Cokic

Hydroxyurea induces fetal hemoglobin by the nitric oxide–dependent activation of soluble guanylyl cyclase

Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of gamma-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced gamma-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.

Erythropoietin and hypoxia increase erythropoietin receptor and nitric oxide levels in lung microvascular endothelial cells

Acute lung exposure to low oxygen results in pulmonary vasoconstriction and redistribution of blood flow. We used human microvascular endothelial cells from lung (HMVEC-L) to study the acute response to oxygen stress. We observed that hypoxia and erythropoietin (EPO) increased erythropoietin receptor (EPOR) gene expression and protein level in HMVEC-L. In addition, EPO dose- and time-dependently stimulated nitric oxide (NO) production. This NO stimulation was evident despite hypoxia induced reduction of endothelial NO synthase (eNOS) gene expression. Western blot of phospho-eNOS (serine1177) and eNOS and was significantly induced by hypoxia but not after EPO treatment. However, iNOS increased at hypoxia and with EPO stimulation compared to normal oxygen tension. In accordance with our previous results of NO induction by EPO at low oxygen tension in human umbilical vein endothelial cells and bone marrow endothelial cells, these results provide further evidence in HMVEC-L for EPO regulation of NO production to modify the effects of hypoxia and cause compensatory vasoconstriction.

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34+ cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34+ cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs.

JAK-STAT and AKT pathway-coupled genes in erythroid progenitor cells through ontogeny.

BackgroundIt has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival have also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34+ progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny.MethodsHematopoietic CD34+ progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells.ResultsIn microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS2, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes are differentially expressed only in some stages of ontogeny (STATs, GRB2, CREBB). Beside the continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH).ConclusionsThis broad overview of gene expression in erythropoiesis revealed transcription factors differentially expressed in some stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway-coupled NFKBIA and YWHAH genes.

Effect of Wine Polyphenol Resveratrol on the Contractions Elicited Electrically or by Norepinephrine in the Rat Portal Vein

We investigated the effects of resveratrol on rat portal vein (RPV) contractility without endothelium. Contractions were produced by electrical field stimulation of perivascular nerves (EFS), norepinephrine (NE), adenosine 5′‐triphosphate (ATP), high K+ solution and by calcium chloride (CaCl2) in Ca2+‐free and high K+, Ca2+‐free solution. The EFS‐evoked contractions were more sensitive to resveratrol and to NS1619‐selective openers of big calcium‐sensitive (BKCa) channels, than NE‐evoked contractions. Effects of resveratrol on the ATP‐evoked contractions were weak. Blockers of BKCa channels partly inhibited the effect of resveratrol only in EFS‐contracted preparations. Western blotting showed that RPV expressed KCa1.1 protein. Inhibitors of ATP‐ and voltage‐sensitive K+ channels did not modify the effects of resveratrol. None of the antagonists of K+ channels affected the resveratrol inhibition of NE‐evoked contractions and effect of high concentrations of resveratrol on the EFS‐evoked contractions. Resveratrol more potently inhibited CaCl2 than potassium chloride contractions of RPV. Thus, BKCa channels partly mediate the inhibitory effect of resveratrol on the neurogenic contractions of RPV. The smooth muscle Ca2+ channels and/or Ca2+ mobilizing through cells might be involved in the effects of resveratrol on the contractility of RPV. Our results are important for better understanding the impact of resveratrol on the portal circulation. Copyright

Microarray and Proteomic Analyses of Myeloproliferative Neoplasms with a Highlight on the mTOR Signaling Pathway

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34+ cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34+ cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34+ cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34+ cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34+ cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34+ cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling.

The Different Effects of Resveratrol and Naringenin on Isolated Human Umbilical Vein: The Role of ATP‐Sensitive K+ Channels

The blood flow from the placenta to the fetus depends on human umbilical vein (HUV) vascular tone. ATP‐sensitive K+ (KATP) channels link the metabolic state of the cell to membrane potential, and their activation in the HUV represents protection against hypoxia. The aims of our study were to assess the effects of resveratrol and naringenin on the HUV and to define the roles of KATP channels in their effects. Serotonin or 100u2009mMu2009K+ were used for precontraction of the HUV without endothelium. The cumulative concentration–response curves were obtained by adding increasing concentrations of resveratrol or naringenin. Glibenclamide was used, in order to test the role of KATP channels in its effect. Resveratrol induced more potent vasodilatation of serotonin‐ and 100u2009mMu2009K+‐precontracted HUV than naringenin. Glibenclamide induced significant shift to the right of the concentration–response curves of resveratrol and P1075 (a specific opener of KATP channels). Western blotting showed that HUV expressed protein Kir6.1. Thus, resveratrol and naringenin produce dilatation of HUV. It seems that KATP channels are involved in the relaxation of HUV induced by resveratrol, while naringenin seems to interact with other ion channels. The K+ channel‐independent mechanism(s) of these polyphenols could not be excluded. Copyright

The effects of potassium channel opener P1075 on the human saphenous vein and human internal mammary artery.

Because adrenergic contractions can contribute to the development of life-threatening spasm of coronary artery bypass graft, this study was performed to investigate the effect of adenosine 3-phosphate (ATP)-sensitive K+ channel (KATP) opener P1075 on contractions of isolated human saphenous vein (HSV) and human internal mammary artery (HIMA). Phasic contractions were evoked by electric field stimulation (20 Hz) and noradrenaline. The sustained contractions were evoked by phenylephrine. The presence of pore-forming Kir6.1 and Kir6.2 subunits of the KATP channels in the HIMA and only Kir6.2 in the HSV was confirmed immunomorphologically. P1075 inhibited in the HSV only, the electrical field stimulation contractions more strongly than noradrenaline contractions. In addition, the phenylephrine contractions of HSV were more sensitive to P1075 in comparison to those of HIMA. Glibenclamide, a KATP channel blocker antagonized the vasodilatation produced by P1075 in both grafts differently, because its effect was more prominent on the P1075-induced inhibition of contractions of HSV than of HIMA. We conclude that P1075 has a vasorelaxant effect and inhibited adrenergic contractions of the tested grafts. This effect is graft and vasoconstrictor selective and seems to be mediated by Kir6.1- and/or Kir6.2-containing KATP channels. Thus, P1075 can be considered as a potential drug in the prevention of graft spasm.

Angiogenic factors are increased in circulating granulocytes and CD34+ cells of myeloproliferative neoplasms

It has been shown that angiogenesis and inflammation play an important role in development of most hematological malignancies including the myeloproliferative neoplasm (MPN). The aim of this study was to investigate and correlate the levels of key angiogenic molecules such as hypoxia‐inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in peripheral blood and bone marrow cells of MPN patients, along with JAK2V617F mutation allele burden and effects of therapy. HIF‐1α and VEGF gene expression were decreased, while eNOS mRNA levels were increased in granulocytes of MPN patients. Furthermore, positively correlated and increased VEGF and eNOS protein levels were in negative correlation with HIF‐1α levels in granulocytes of MPN patients. According to immunoblotting, the generally augmented angiogenic factors demonstrated JAK2V617F allele burden dependence only in granulocytes of PMF. The angiogenic factors were largely reduced after hydroxyurea therapy in granulocytes of MPN patients. Levels of eNOS protein expression were stimulated by Calreticulin mutations in granulocytes of essential thrombocythemia. Immunocytochemical analyses of CD34+ cells showed a more pronounced enhancement of angiogenic factors than in granulocytes. Increased gene expression linked to the proinflammatory TGFβ and MAPK signaling pathways were detected in CD34+ cells of MPN patients. In conclusion, the angiogenesis is increased in several cell types of MPN patients supported by the transcriptional activation of inflammation‐related target genes, and is not limited to bone marrow stroma cells. It also appears that some of the benefit of hydroxyurea therapy of the MPN is mediated by effects on angiogenic factors.

Stimulated stromal cells induce γ-globin gene expression in erythroid cells via nitric oxide production

OBJECTIVEnWe have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). We have now expanded those studies to demonstrate that stimulation of gamma-globin gene expression is also mediated by NOS induction in stromal cells within the bone marrow microenvironment.nnnMATERIALS AND METHODSnUsing NO analyzer, we measured NO production in endothelial and macrophage cell cultures. In coculture studies of erythroid and stromal cells, we measured globin gene expression during stimulation by NO inducers.nnnRESULTSnHydroxyurea (30-100 microM) induced NOS-dependent production of NO in human macrophages (up to 1.2 microM). Coculture studies of human macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to threefold) in the presence of hydroxyurea. NOS-dependent stimulation of NO by lipopolysaccharide (up to 0.6 microM) has been observed in human macrophages. We found that lipopolysaccharide and interferon-gamma together increased gamma-globin gene expression (up to twofold) in human macrophage/erythroid cell cocultures. Coculture of human bone marrow endothelial cells with erythroid progenitor cells also induced gamma-globin mRNA expression (2.4-fold) in the presence of hydroxyurea (40 microM).nnnCONCLUSIONnThese results demonstrate an arrangement by which NO and fetal hemoglobin inducers may stimulate globin genes in erythroid cells via the common paracrine effect of bone marrow stromal cells.