RESUMO
Chronic inflammation has been identified in leukemias as an essential regulator of angiogenesis. B-chronic lymphocytic leukemia (CLL) cells secrete high levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 alpha (HIF1α). The aim was to assess the role of inflammation in activation of angiogenic factors: endothelial nitric oxide synthase (eNOS), HIF1α and VEGF via proliferation related signaling pathways and VEGF autocrine control. We isolated mononuclear cells (MNC) and CD19+ cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-κB (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR. Also, we investigated IL-6 dependent neovascularization in human microvascular endothelial cells (HMEC-1) in co-culture with MNC of CLL. The angiogenic factors eNOS, VEGF and HIF1α had significantly higher frequencies in MNC of CLL in comparison to healthy controls (p < 0.001) and CD19+ cells of CLL. IL-6 increased the quantity of HIF1α (p < 0.05) and VEGF positive cells in the presence of JSH23 (p < 0.01). VEGF increased HIF1α (p < 0.05), and decreased eNOS gene expression (p < 0.01) in MNC of CLL. VEGF significantly (p < 0.001) increased the number of HIF1α positive MNC of CLL, prevented by inhibitors of JAK1/2, PI3K and mTOR signaling pathways. VEGF stimulation of SMAD (p < 0.05) and STAT5 (p < 0.01) signaling has been prevented by inhibitors of JAK1/2, mTOR, PI3K and SMAD signaling, individually (p < 0.01) or mutually (p < 0.001). Also, we showed that MNC of CLL and IL-6 individually stimulate neovascularization in co-culture with HMEC-1, without a cumulative effect. We demonstrated elevated angiogenic factors in CLL, while VEGF and IL-6 independently stimulated HIF1α. VEGF stimulation of HIF1α was mostly mTOR dependent, while IL-6 stimulation was NF-κB dependent.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Inflamação , Leucemia Linfocítica Crônica de Células B , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Neovascularização Patológica/metabolismo , Feminino , Masculino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Idoso , Pessoa de Meia-Idade , Inflamação/metabolismo , Transdução de Sinais , Interleucina-6/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Idoso de 80 Anos ou mais , Adulto , AngiogêneseRESUMO
Neutrophils are an essential component of the innate immune response, but their prolonged activation can lead to chronic inflammation. Consequently, neutrophil homeostasis is tightly regulated through balance between granulopoiesis and clearance of dying cells. The bone marrow is both a site of neutrophil production and the place they return to and die. Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by the mutations in three types of molecular markers, with emphasis on Janus kinase 2 gene mutation (JAK2V617F). The MPN bone marrow stem cell niche is a site of chronic inflammation, with commonly increased cells of myeloid lineage, including neutrophils. The MPN neutrophils are characterized by the upregulation of JAK target genes. Additionally, MPN neutrophils display malignant nature, they are in a state of activation, and with deregulated apoptotic machinery. In other words, neutrophils deserve to be placed in the midst of major events in MPN. Our crucial interest in this review is better understanding of how neutrophils die in MPN mirrored by defects in apoptosis and to what possible extent they can contribute to MPN pathophysiology. We tend to expect that reduced neutrophil apoptosis will establish a pathogenic link to chronic inflammation in MPN.
Assuntos
Neoplasias Hematológicas/imunologia , Imunidade Inata , Transtornos Mieloproliferativos/imunologia , Neutrófilos/imunologia , Substituição de Aminoácidos , Animais , Doença Crônica , Neoplasias Hematológicas/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/genéticaRESUMO
Increased energy requirement and metabolic reprogramming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2-driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells, resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy, and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high-fat diet improved survival, whereas high-glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPNs. These effects were additive with the JAK1/2 inhibitor ruxolitinib in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPNs and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPNs.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Janus Quinase 2/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Animais , Humanos , Camundongos , MutaçãoRESUMO
This study has been performed to determine the mechanism of activation of the myeloid related S100A proteins by inflammatory cytokines in myeloproliferative neoplasm (MPN). Besides microarray analysis of MPN-derived CD34+ cells, we analysed the pro-inflammatory IL6 and anti-inflammatory IL10 dependence of NF-κB, PI3K-AKT, and JAK-STAT signalling during induction of S100A proteins in mononuclear cells of MPN, by immunoblotting and flow cytometry. We observed the reduced gene expression linked to NF-κB and inflammation signalling in MPN-derived CD34+ cells. Both IL6 and IL10 reduced S100A8 and 100A9 protein levels mediated via NF-κB and PI3K signalling, respectively, in mononuclear cells of essential thrombocythemia (ET). We also determined the increased percentage of S100A8 and S100A9 positive granulocytes in ET and primary myelofibrosis, upgraded by the JAK2V617F mutant allele burden. S100A8/9 heterodimer induced JAK1/2-dependent mitotic arrest of the ET-derived granulocytes. SIGNIFICANCE OF THE STUDY: We demonstrated that inflammation reduced the myeloid related S100A8/9 proteins by negative feedback mechanism in ET. S100A8/9 can be a diagnostic marker of inflammation in MPN, supported by the concomitant NF-κB and JAK1/2 signalling inhibition in regulation of myeloproliferation and therapy of MPN.
Assuntos
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Interleucina-6/metabolismo , Leucócitos Mononucleares/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Trombocitemia Essencial/metabolismo , Substituição de Aminoácidos , Calgranulina A/genética , Calgranulina B/genética , Feminino , Humanos , Interleucina-6/genética , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Mutação de Sentido Incorreto , NF-kappa B/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Trombocitemia Essencial/genética , Trombocitemia Essencial/patologiaRESUMO
Myeloproliferative neoplasms (MPNs) are developing resistance to therapy by JAK1/2 inhibitor ruxolitinib. To explore the mechanism of ruxolitinib's limited effect, we examined the JAK1/2 mediated induction of proliferation related ERK1/2 and AKT signaling by proinflammatory interleukin-6 (IL-6) in MPN granulocytes and JAK2V617F mutated human erythroleukemia (HEL) cells. We found that JAK1/2 or JAK2 inhibition prevented the IL-6 activation of STAT3 and AKT pathways in polycythemia vera and HEL cells. Further, we showed that these inhibitors also blocked the IL-6 activation of the AKT pathway in primary myelofibrosis (PMF). Only JAK1/2 inhibitor ruxolitinib largely activated ERK1/2 signaling in essential thrombocythemia and PMF (up to 4.6 fold), with a more prominent activation in JAK2V617F positive granulocytes. Regarding a cell cycle, we found that IL-6 reduction of HEL cells percentage in G2M phase was reversed by ruxolitinib (2.6 fold). Moreover, ruxolitinib potentiated apoptosis of PMF granulocytes (1.6 fold). Regarding DNA replication, we found that ruxolitinib prevented the IL-6 augmentation of MPN granulocytes frequency in the S phase of the cell cycle (up to 2.9 fold). The inflammatory stimulation induces a cross-talk between the proliferation linked pathways, where JAK1/2 inhibition is compensated by the activation of the ERK1/2 pathway during IL-6 stimulation of DNA replication.
Assuntos
Replicação do DNA/efeitos dos fármacos , Interleucina-6/farmacologia , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Transtornos Mieloproliferativos/patologia , Adulto , Idoso , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Feminino , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/metabolismo , Nitrilas , Fosforilação/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único , Pirazóis/farmacologia , Pirimidinas , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Fatores de Transcrição STAT/metabolismoRESUMO
In accordance with increased proliferation in myeloproliferative neoplasm (MPN), the goal is to evaluate the immunoexpression of: ß-catenin, PPAR-γ and Ki67 protein, to compare them with bone marrow ultrastructural characteristics in patients with MPN. Immunoexpression and electron microscopy of bone marrow was analyzed in 30 Ph-negative MPN patients, including per 10 patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The quantity of ß-catenin immunoreactive cells was significantly higher in PV then in ET (p < 0.01) or PMF group of patients (p < 0.01) and also in ET versus PMF group of patients (p < 0.01). Erythroid lineage showed absent ß-catenin staining without immunoreactivity in nucleus. In contrast, immunoreactivity for PPAR-γ was localized mostly in megakaryocytes and the highest number of PPAR-γ immunopositive cells was detected in PMF group of patients. In addition, the proliferative Ki67 index was significantly increased in the PMF and PV patients compared to patients with ET. Also, the megakaryocytes showed abnormal maturation in PMF group of patients as determined by ultrastructural analysis. These results indicated that PV dominantly expressed ß-catenin and proliferation marker Ki67 in bone marrow, while PMF is linked preferentially to PPAR-γ immunopositive megakaryocytes characterized by abnormal maturation.
Assuntos
Medula Óssea/metabolismo , Transtornos Mieloproliferativos/metabolismo , PPAR gama/metabolismo , beta Catenina/metabolismo , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Megacariócitos/metabolismo , Pessoa de Meia-Idade , Policitemia Vera/metabolismo , Mielofibrose Primária/metabolismoRESUMO
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 TGFß 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. © 2016 Wiley Periodicals, Inc.
Assuntos
Antígenos CD34/análise , Medula Óssea/patologia , Granulócitos/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/sangue , Transtornos Mieloproliferativos/sangue , Óxido Nítrico Sintase Tipo III/sangue , Fator A de Crescimento do Endotélio Vascular/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Calreticulina/genética , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Neovascularização Patológica/sangue , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Óxido Nítrico Sintase Tipo III/análise , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
Increased angiogenesis in BCR-ABL1 negative myeloproliferative neoplasms (MPNs) has been recognized, but its connection with clinical and molecular markers needs to be defined. The aims of study were to (1) assess bone marrow (BM) angiogenesis measured by microvessel density (MVD) using CD34 and CD105 antibodies; (2) analyze correlation of MVD with plasma angiogenic factors including vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8; (3) examine the association of MVD with clinicopathological and molecular markers. We examined 90 de novo MPN patients (30 polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET)) and 10 age-matched controls. MVD was analyzed by immunohistochemistry "hot spot" method, angiogenic factors by immunoassay and JAK2V617F, and CALR mutations by DNA sequencing and allelic PCR. MVD was significantly increased in MPNs compared to controls (PMF > PV > ET). Correlation between MVD and plasma angiogenic factors was found in MPNs. MVD was significantly increased in patients with JAK2V617F mutation and correlated with JAK2 mutant allele burden (CD34-MVD: ρ = 0.491, p < 0.001; CD105-MVD: ρ = 0.276, p = 0.02) but not with CALR mutation. MVD correlated with leukocyte count, serum lactate dehydrogenase, hepatomegaly, and splenomegaly. BM fibrosis was significantly associated with CD34-MVD, CD105-MVD, interleukin-8, and JAK2 mutant allele burden. JAK2 homozygote status had positive predictive value (100%) for BM fibrosis. Patients with prefibrotic PMF had significantly higher MVD than patients with ET, and we could recommend MVD to be additional histopathological marker to distinguish these two entities. This study also highlights the strong correlation of MVD with plasma angiogenic factors, JAK2 mutant allele burden, and BM fibrosis in MPNs.
Assuntos
Indutores da Angiogênese/sangue , Medula Óssea/irrigação sanguínea , Medula Óssea/patologia , Microvasos/patologia , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/patologia , Adulto , Idoso , Medula Óssea/metabolismo , Feminino , Humanos , Masculino , Microvasos/metabolismo , Pessoa de Meia-Idade , Neovascularização Patológica/sangue , Neovascularização Patológica/patologia , Estudos ProspectivosRESUMO
PURPOSE: We compared the gene expression profile of peripheral blood CD34(+) cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR-ABL oncogene. METHODS: The microarray analyses have been performed in circulating CD34(+) cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR-ABL positive CML patients were in chronic phase, with a mean value of 2012±SD of CD34(+)cells/µl in peripheral blood. RESULTS: The gene expression profile was more prominent in CML CD34(+) cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34(+) cells (HINT1, TXN, SERBP1) and granulocytes, respectively. BCR-ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11, CDK4/6, and MYC and reduction of E2F1, KRAS, and NFKBIA gene expression in CD34(+) cells. Among genes linked to the inhibition of cellular proliferation by BCR-ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. CONCLUSION: The presence of BCR-ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34(+) cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects.
Assuntos
Antígenos CD34/metabolismo , Regulação Leucêmica da Expressão Gênica , Granulócitos/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Células-Tronco Neoplásicas/metabolismo , Transcriptoma , Biomarcadores , Estudos de Casos e Controles , Análise por Conglomerados , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Perfilação da Expressão Gênica , Granulócitos/patologia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/patologia , Transdução de SinaisRESUMO
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.
Assuntos
Interleucina-6/sangue , Janus Quinase 1/sangue , Transtornos Mieloproliferativos/imunologia , Fatores de Transcrição STAT/sangue , Alelos , Antígenos CD34/metabolismo , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Inflamação , Leucocitose/complicações , Masculino , Mutação , Transtornos Mieloproliferativos/sangue , Análise de Sequência com Séries de Oligonucleotídeos , Policitemia Vera/sangue , Policitemia Vera/imunologia , Mielofibrose Primária/sangue , Mielofibrose Primária/imunologia , Análise de Sequência de DNA , Transdução de Sinais , Trombocitemia Essencial/sangue , Trombocitemia Essencial/imunologia , Trombocitose/sangue , Trombocitose/imunologiaRESUMO
Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 µM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 µM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions.
Assuntos
Hipóxia Celular/fisiologia , Células Endoteliais/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Receptores da Eritropoetina/metabolismo , Células Endoteliais/efeitos dos fármacos , Eritropoetina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Compostos Nitrosos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Eritropoetina/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: The purpose of this study was to examine the gene expression profile of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood (mPB)-derived progenitors, used in transplantation. METHODS: We correlated gene expression patterns of highly enriched steady-state peripheral blood (PB)- and mPB-derived CD71+ cells by microarray and ingenuity pathway analyses, to identify the transcriptional program during in vitro erythroid differentiation. RESULTS: The gene expression was more than doubled in mPB-derived (4180 genes) compared to PB-derived erythroid progenitors (1667 genes) while PB-and mPB-derived erythroid progenitors shared 1534 common genes. Comparative analysis of transcript levels showed differential expression of 54 genes between cultured erythroid progenitors of PB and mPB origin, where we identified common 13 downregulated and 30 upregulated genes. The most significant genes in mPB-derived erythroid progenitors were P4HB, DDIA3, ARPC2 and ATP5G3. Regarding G-CSF stimulation the G-CSF receptor CSF2RB (1.1-fold) was linked via STAT3 to erythroid-specific ALAS2 (2.9-fold) and GATA2 (1.3-fold) factors, all upregulated in mPB-derived erythroid progenitors, coupled to common upregulated NUDC gene involved in the proliferation of erythroid cells. CONCLUSION: This report provides an extensive transcriptional profile of cultured erythroid progenitors and leads to a better understanding of diversity among the progenitor sources.
Assuntos
Células Precursoras Eritroides/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Transcriptoma , Diferenciação Celular/genética , Células Cultivadas , Células Precursoras Eritroides/transplante , Redes Reguladoras de Genes/genética , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Análise Serial de TecidosRESUMO
BACKGROUND: The guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) regulate cell growth, proliferation and differentiation. G proteins are also implicated in erythroid differentiation, and some of them are expressed principally in hematopoietic cells. GPCRs-linked NO/cGMP and p38 MAPK signaling pathways already demonstrated potency for globin gene stimulation. By analyzing erythroid progenitors, derived from hematopoietic cells through in vitro ontogeny, our study intends to determine early markers and signaling pathways of globin gene regulation and their relation to GPCR expression. RESULTS: Human hematopoietic CD34+ progenitors are isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and G-CSF stimulated mobilized PB (mPB), and then differentiated in vitro into erythroid progenitors. We find that growth capacity is most abundant in FL- and CB-derived erythroid cells. The erythroid progenitor cells are sorted as 100% CD71+, but we did not find statistical significance in the variations of CD34, CD36 and GlyA antigens and that confirms similarity in maturation of studied ontogenic periods. During ontogeny, beta-globin gene expression reaches maximum levels in cells of adult blood origin (176 fmol/µg), while gamma-globin gene expression is consistently up-regulated in CB-derived cells (60 fmol/µg). During gamma-globin induction by hydroxycarbamide, we identify stimulated GPCRs (PTGDR, PTGER1) and GPCRs-coupled genes known to be activated via the cAMP/PKA (ADIPOQ), MAPK pathway (JUN) and NO/cGMP (PRPF18) signaling pathways. During ontogeny, GPR45 and ARRDC1 genes have the most prominent expression in FL-derived erythroid progenitor cells, GNL3 and GRP65 genes in CB-derived cells (high gamma-globin gene expression), GPR110 and GNG10 in BM-derived cells, GPR89C and GPR172A in PB-derived cells, and GPR44 and GNAQ genes in mPB-derived cells (high beta-globin gene expression). CONCLUSIONS: These results demonstrate the concomitant activity of GPCR-coupled genes and related signaling pathways during erythropoietic stimulation of globin genes. In accordance with previous reports, the stimulation of GPCRs supports the postulated connection between cAMP/PKA and NO/cGMP pathways in activation of γ-globin expression, via JUN and p38 MAPK signaling.
Assuntos
Diferenciação Celular , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Proteínas de Ligação ao GTP/genética , gama-Globinas/genética , Proliferação de Células , Células Precursoras Eritroides/citologia , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Globinas beta/genética , Globinas beta/metabolismo , gama-Globinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
AIMS: We showed previously that the acute effect of ethanol on intestinal immunoglobulin A (IgA) expression might be mediated by endogenous nitric oxide (NO). To extend these findings, this study was designed to investigate a possible role of neuronal NO synthase (nNOS) in the observed phenomenon, using 7-nitroindazole (7-NI), a selective inhibitor of its activity. METHODS: Adult male Wistar rats were treated with: (a) ethanol (4 g/kg, intraperitoneally, i.p.), (b) 7-NI (25 mg/kg, i.p.) followed by ethanol (4 g/kg, i.p.) 30 min later and (c) 7-NI (25 mg/kg, i.p.) followed by saline 30 min later. Untreated rats were used as controls. The concentrations of serum and intestinal IgA were measured by enzyme-linked immunosorbent assay, while the expression of nNOS was determined using western blot and immunohistochemistry. RESULTS: Acute ethanol treatment significantly increased the concentration of IgA in the ileal extracts, whereas it decreased its serum level. Inhibition of nNOS activity by 7-NI abolished this action of alcohol on IgA. Additionally, western blot analysis revealed that the acute alcohol administration induced an increase in the expression of intestinal nNOS. Furthermore, nNOS-immunoreactive cells, observed within the lamina propria of small intestine, were numerous in ethanol-treated rats. CONCLUSION: Taken together, these results extended our previous findings suggesting that nNOS mediates the acute effect of ethanol on IgA and supported an immunomodulatory role of this enzyme isoform.
Assuntos
Etanol/toxicidade , Imunoglobulina A/metabolismo , Óxido Nítrico Sintase Tipo I/fisiologia , Animais , Biomarcadores/sangue , Íleo/efeitos dos fármacos , Íleo/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
The severity and mortality of coronavirus disease 2019 (COVID-19) are greater in males than in females, though the infection rate is the same in the two sexes. We investigated sex hormone differences associated with the hyperinflammatory immune response to SARS-CoV-2 on the basis of patients' cytokine profiles and vaccination statuses. Clinical and laboratory data of 117 patients with COVID-19 were collected to examine sex differences associated with oxidative stress markers, neutrophil extracellular traps (NETs), and plasma cytokine levels up to 5 months from hospital admission. The testosterone and free testosterone levels were low in male patients with COVID-19 and returned to normal values after recovery from the disease. The dihydrotestosterone (DHT) levels were transiently reduced, while the sex hormone-binding globulin levels were decreased in post-COVID-19 male patients. The levels of the inflammatory cytokines interleukin-6 (IL-6) and IL-10 appeared generally increased at diagnosis and decreased in post-COVID-19 patients. In females, the concentration of tumor necrosis factor-alpha was increased by four times at diagnosis. The levels of the coagulation markers intercellular adhesion molecule-1 (ICAM-1) and E-selectin were consistently upregulated in post-COVID-19 female patients, in contrast to those of vascular cell adhesion molecule-1 (VCAM-1), P-selectin, and chemokine IL-8. DHT increased the levels of reactive oxygen species in the neutrophils of male patients, while estradiol decreased them in females. Markers for NET, such as circulating DNA and myeloperoxidase, were significantly more abundant in the patients' plasma. Sex hormones have a potential protective role during SARS-CoV-2 infection, which is weakened by impaired testosterone synthesis in men.
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BACKGROUND: It 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. METHODS: Hematopoietic 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. RESULTS: In 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). CONCLUSIONS: This 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.
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Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/enzimologia , Janus Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Transcrição STAT/genética , Transdução de Sinais/genética , Adulto , Proliferação de Células , Sobrevivência Celular/genética , Análise por Conglomerados , Células Precursoras Eritroides/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hematopoese/genética , Humanos , Janus Quinases/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição STAT/metabolismoRESUMO
The COVID-19 pandemic has had a strong impact on people's quality of life (QoL), which is affected by social and economic changes as well as by mental and physical health. The aim of this study was to determine QoL in post-COVID-19 patients who had required hospitalization, and to identify relevant sociodemographic data. We used questionnaires which considered demographic and socioeconomic data, health and vaccination status, the pandemic situation, and EQ-5D scoring. The interactions of all data and the scores of EQ-5D were analyzed. Multivariate logistic regression analysis was applied to the five dimensions of EQ-5D. In this single-hospital-cohort study, the average times elapsed since initial diagnosis and hospital admission were 2.5 (76.3 ± 18.1 days) and 5 months (155.4 ± 33.9 days), respectively. Post-COVID-19 females were 3-5 times more likely to be affected in terms of anxiety/depression, and in negative impact upon their usual activities, at 5 months after diagnosis. At the same time, reductions in mobility were 3-4 times more likely in elderly post-COVID-19 patients, whose levels of pain and discomfort increased. Single patients, those with low incomes, and those with severe clinical outcomes were 2-4 times more likely to experience a reduction in their usual activities, while the presence of co-morbidities and lower levels of education were associated with increased pain and discomfort. Aging-induced pain/discomfort and anxiety/depression were significantly exacerbated in elderly patients with widespread vaccination. Our study revealed effects of demographic and socioeconomic factors upon lower QoL in post-COVID-19 patients in four dimensions of EQ-5D: mobility, usual activity, pain/discomfort, and anxiety/depression, 5 months after first diagnosis and hospitalization.
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Although bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been identified as a major cellular source of fibrosis, the exact molecular mechanism and signaling pathways involved have not been identified thus far. Here, we show that BM-MSCs contribute to fibrosis in myeloproliferative neoplasms (MPNs) by differentiating into αSMA-positive myofibroblasts. These cells display a dysregulated extracellular matrix with increased FN1 production and secretion of profibrotic MMP9 compared to healthy donor cells. Fibrogenic TGFß and inflammatory JAK2/STAT3 and NFκB signaling pathway activity is increased in BM-MSCs of MPN patients. Moreover, coculture with mononuclear cells from MPN patients was sufficient to induce fibrosis in healthy BM-MSCs. Inhibition of JAK1/2, SMAD3 or NFκB significantly reduced the fibrotic phenotype of MPN BM-MSCs and was able to prevent the development of fibrosis induced by coculture of healthy BM-MSCs and MPN mononuclear cells with overly active JAK/STAT signaling, underlining their involvement in fibrosis. Combined treatment with JAK1/2 and SMAD3 inhibitors showed synergistic and the most favorable effects on αSMA and FN1 expression in BM-MSCs. These results support the combined inhibition of TGFß and inflammatory signaling to extenuate fibrosis in MPN.
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Células-Tronco Mesenquimais , Neoplasias , Medula Óssea/metabolismo , Medula Óssea/patologia , Células da Medula Óssea/metabolismo , Fibrose , Humanos , Células-Tronco Mesenquimais/metabolismo , Neoplasias/metabolismo , Transdução de SinaisRESUMO
Chronic inflammation is characterized by the production of reactive oxygen species (ROS), reactive nitrogen species, and inflammatory cytokines in myeloproliferative neoplasms (MPNs). In addition to these parameters, the aim of this study was to analyze the influence of ROS on the proliferation-related AKT/mTOR signaling pathway and the relationship with inflammatory factors in chronic myelogenous leukemia (CML). The activity of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase is reduced in erythrocytes while levels of the oxidative stress markers malondialdehyde and protein carbonyl are elevated in the plasma of patients with CML. In addition, nitrogen species (nitrotyrosine, iNOS, eNOS) and inflammation markers (IL-6, NFkB, and S100 protein) were increased in granulocytes of CML while anti-inflammatory levels of IL-10 were decreased in plasma. CML granulocytes exhibited greater resistance to cytotoxic H2O2 activity compared to healthy subjects. Moreover, phosphorylation of the apoptotic p53 protein was reduced while the activity of the AKT/mTOR signaling pathway was increased, which was further enhanced by oxidative stress (H2O2) in granulocytes and erythroleukemic K562 cells. IL-6 caused oxidative stress and DNA damage that was mitigated using antioxidant or inhibition of inflammatory NFkB transcription factor in K562 cells. We demonstrated the presence of oxidative and nitrosative stress in CML, with the former mediated by AKT/mTOR signaling and stimulated by inflammation.
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Peróxido de Hidrogênio , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Peróxido de Hidrogênio/farmacologia , Inflamação , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Estresse Nitrosativo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.