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1.
Front Pharmacol ; 12: 725734, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34603036

RESUMO

Erythropoietin (EPO) receptor (EPOR) determines EPO response. High level EPOR on erythroid progenitor cells gives rise to EPO regulated production of red blood cells. Animal models provide evidence for EPO activity in non-hematopoietic tissue mediated by EPOR expression. Beyond erythropoiesis, EPO activity includes neuroprotection in brain ischemia and trauma, endothelial nitric oxide production and cardioprotection, skeletal muscle wound healing, and context dependent bone remodeling affecting bone repair or bone loss. This review highlights examples of EPO protective activity in select non-hematopoietic tissue with emphasis on metabolic response mediated by EPOR expression in fat and brain and sex-specific regulation of fat mass and inflammation associated with diet induced obesity. Endogenous EPO maintains glucose and insulin tolerance and protects against fat mass accumulation and inflammation. Accompanying the increase in erythropoiesis with EPO treatment is improved glucose tolerance and insulin response. During high fat diet feeding, EPO also decreases fat mass accumulation in male mice. The increased white adipose tissue inflammation and macrophage infiltration associated with diet induced obesity are also reduced with EPO treatment with a shift toward an anti-inflammatory state and decreased inflammatory cytokine production. In female mice the protective effect of estrogen against obesity supersedes EPO regulation of fat mass and inflammation, and requires estrogen receptor alpha activity. In brain, EPOR expression in the hypothalamus localizes to proopiomelanocortin neurons in the arcuate nucleus that promotes a lean phenotype. EPO stimulation of proopiomelanocortin neurons increases STAT3 signaling and production of proopiomelanocortin. Cerebral EPO contributes to metabolic response, and elevated brain EPO reduces fat mass and hypothalamus inflammation during diet induced obesity in male mice without affecting EPO stimulated erythropoiesis. Ovariectomy abrogates the sex-specific metabolic response of brain EPO. The sex-dimorphic EPO metabolic response associated with fat mass accumulation and inflammation during diet induced obesity provide evidence for crosstalk between estrogen and EPO in their anti-obesity potential in female mice mediated in part via tissue specific response in brain and white adipose tissue. Endogenous and exogenous EPO response in non-hematopoietic tissue demonstrated in animal models suggests additional activity by which EPO treatment may affect human health beyond increased erythropoiesis.

2.
J Bone Miner Res ; 36(7): 1211-1219, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33949002

RESUMO

Erythropoietin (EPO) is the primary regulator of bone marrow erythropoiesis. Mouse models have provided evidence that EPO also promotes bone remodeling and that EPO-stimulated erythropoiesis is accompanied by bone loss independent of increased red blood cell production. EPO has been used clinically for three decades to treat anemia in end-stage renal disease, and notably, although the incidence of hip fractures decreased in the United States generally after 1990, it rose among hemodialysis patients coincident with the introduction and subsequent dose escalation of EPO treatment. Given this clinical paradox and findings from studies in mice that elevated EPO affects bone health, we examined EPO treatment as a risk factor for fractures in hemodialysis patients. Relationships between EPO treatment and hip fractures were analyzed using United States Renal Data System (USRDS) datasets from 1997 to 2013 and Consolidated Renal Operations in a Web-enabled Network (CROWNWeb) datasets for 2013. Fracture risks for patients treated with <50 units of EPO/kg/week were compared to those receiving higher doses by multivariable Cox regression. Hip fracture rates for 747,832 patients in USRDS datasets (1997-2013) increased from 12.0 per 1000 patient years in 1997 to 18.9 in 2004, then decreased to 13.1 by 2013. Concomitantly, average EPO doses increased from 11,900 units/week in 1997 to 18,300 in 2004, then decreased to 8,800 by 2013. During this time, adjusted hazard ratios for hip fractures with EPO doses of 50-149, 150-299, and ≥ 300 units/kg/week compared to <50 units/kg/week were 1.08 (95% confidence interval [CI], 1.01-1.15), 1.22 (95% CI, 1.14-1.31), and 1.41 (95% CI, 1.31-1.52), respectively. Multivariable analyses of 128,941 patients in CROWNWeb datasets (2013) replicated these findings. This study implicates EPO treatment as an independent risk factor for hip fractures in hemodialysis patients and supports the conclusion that EPO treatment may have contributed to changing trends in fracture incidence for these patients during recent decades. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).


Assuntos
Anemia , Eritropoetina , Fraturas do Quadril , Falência Renal Crônica , Anemia/complicações , Anemia/tratamento farmacológico , Anemia/epidemiologia , Animais , Fraturas do Quadril/epidemiologia , Humanos , Falência Renal Crônica/complicações , Falência Renal Crônica/terapia , Camundongos , Diálise Renal , Estados Unidos/epidemiologia
3.
FASEB J ; 34(9): 11685-11697, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32671900

RESUMO

Erythropoietin (EPO) regulates erythropoiesis by binding to erythropoietin receptor (Epor) on erythroid progenitor cells. Epor is also expressed on bone forming osteoblasts and bone loss accompanies EPO-stimulated erythropoiesis in mice. Mice with Epor restricted to erythroid tissue exhibit reduced bone and increased marrow adipocytes; in contrast, transgenic mice (Tg) with osteoblastic-specific deletion of Epor exhibit reduced trabecular bone with age without change in marrow adipocytes. By 12 weeks, male Tg mice had 22.2% and female Tg mice had 29.6% reduced trabecular bone volume (BV) compared to controls. EPO administration (1200 U/kg) for 10 days reduced trabecular bone in control mice but not in Tg mice. There were no differences in numbers of osteoblasts, osteoclasts, and marrow adipocytes in Tg mice, suggesting independence of EPO signaling in mature osteoblasts, osteoclasts, and adipocytes. Female Tg mice had increased number of dying osteocytes and male Tg mice had a trend for more empty lacunae. Osteogenic cultures from Tg mice had reduced differentiation and mineralization with reduced Alpl and Runx2 transcripts. In conclusion, endogenous EPO-Epor signaling in osteoblasts is important in bone remodeling, particularly trabecular bone and endogenous Epor expression in osteoblasts is required for bone loss accompanying EPO-stimulated erythropoiesis.


Assuntos
Eritropoese/efeitos dos fármacos , Eritropoetina/administração & dosagem , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Receptores da Eritropoetina/genética , Animais , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Eritropoese/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osteoblastos/citologia , Osteoclastos/citologia , Osteócitos/metabolismo , Receptores da Eritropoetina/metabolismo , Transdução de Sinais/genética
4.
JCI Insight ; 5(5)2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32078583

RESUMO

The blood hormone erythropoietin (EPO), upon binding to its receptor (EpoR), modulates high-fat diet-induced (HFD-induced) obesity in mice, improves glucose tolerance, and prevents white adipose tissue inflammation. Transgenic mice with constitutive overexpression of human EPO solely in the brain (Tg21) were used to assess the neuroendocrine EPO effect without increasing the hematocrit. Male Tg21 mice resisted HFD-induced weight gain; showed lower serum adrenocorticotropic hormone, corticosterone, and C-reactive protein levels; and prevented myeloid cell recruitment to the hypothalamus compared with WT male mice. HFD-induced hypothalamic inflammation (HI) and microglial activation were higher in male mice, and Tg21 male mice exhibited a lower increase in HI than WT male mice. Physiological EPO function in the brain also showed sexual dimorphism in regulating HFD response. Female estrogen production blocked reduced weight gain and HI. Targeted deletion of EpoR gene expression in neuronal cells worsened HFD-induced glucose intolerance in both male and female mice but increased weight gain and HI in the hypothalamus in male mice only. Both male and female Tg21 mice kept on normal chow and HFD showed significantly improved glycemic control. Our data indicate that cerebral EPO regulates weight gain and HI in a sex-dependent response, distinct from EPO regulation of glycemic control, and independent of erythropoietic EPO response.


Assuntos
Encéfalo/metabolismo , Eritropoetina/metabolismo , Hipotálamo/patologia , Inflamação/metabolismo , Fatores Sexuais , Animais , Glicemia/metabolismo , Comportamento Alimentar , Feminino , Hipotálamo/metabolismo , Resistência à Insulina , Masculino , Camundongos , Camundongos Transgênicos , Receptores da Eritropoetina/genética
5.
Nitric Oxide ; 90: 10-14, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173908

RESUMO

The mechanism for nitric oxide (NO) generation from reduction of nitrate (NO3-) and nitrite (NO2-) has gained increasing attention due to the potential beneficial effects of NO in cardiovascular diseases and exercise performance. We have previously shown in rodents that skeletal muscle is the major nitrate reservoir in the body and that exercise enhances the nitrate reduction pathway in the muscle tissue and have proposed that nitrate in muscle originates from diet, the futile cycle of nitric oxide synthase 1 (NOS1) and/or oxidation of NO by oxymyoglobin. In the present study, we tested the hypothesis that lack of myoglobin expression would decrease nitrate levels in skeletal muscle. We observed a modest but significant decrease of nitrate level in skeletal muscle of myoglobin deficient mice compared to littermate control mice (17.3 vs 12.8 nmol/g). In contrast, a NOS inhibitor, L-NAME or a low nitrite/nitrate diet treatment led to more pronounced decreases of nitrate levels in the skeletal muscle of both control and myoglobin deficient mice. Nitrite levels in the skeletal muscle of both types of mice were similar (0.48 vs 0.42 nmol/g). We also analyzed the expression of several proteins that are closely related to NO metabolism to examine the mechanism by which nitrate and nitrite levels are preserved in the absence of myoglobin. Western blot analyses suggest that the protein levels of xanthine oxidoreductase and sialin, a nitrate transporter, both increased in the skeletal muscle of myoglobin deficient mice. These results are compatible with our previously reported model of nitrate production in muscle and suggest that myoglobin deficiency activates compensatory mechanisms to sustain NO homeostasis.


Assuntos
Homeostase , Mioglobina/deficiência , Mioglobina/metabolismo , Óxido Nítrico/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo
6.
Front Physiol ; 10: 1534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32038269

RESUMO

In mammals, erythropoietin (EPO), produced in the kidney, is essential for bone marrow erythropoiesis, and hypoxia induction of EPO production provides for the important erythropoietic response to ischemic stress, such as during blood loss and at high altitude. Erythropoietin acts by binding to its cell surface receptor which is expressed at the highest level on erythroid progenitor cells to promote cell survival, proliferation, and differentiation in production of mature red blood cells. In addition to bone marrow erythropoiesis, EPO causes multi-tissue responses associated with erythropoietin receptor (EPOR) expression in non-erythroid cells such neural cells, endothelial cells, and skeletal muscle myoblasts. Animal and cell models of ischemic stress have been useful in elucidating the potential benefit of EPO affecting maintenance and repair of several non-hematopoietic organs including brain, heart and skeletal muscle. Metabolic and glucose homeostasis are affected by endogenous EPO and erythropoietin administration affect, in part via EPOR expression in white adipose tissue. In diet-induced obese mice, EPO is protective for white adipose tissue inflammation and gives rise to a gender specific response in weight control associated with white fat mass accumulation. Erythropoietin regulation of fat mass is masked in female mice due to estrogen production. EPOR is also expressed in bone marrow stromal cells (BMSC) and EPO administration in mice results in reduced bone independent of the increase in hematocrit. Concomitant reduction in bone marrow adipocytes and bone morphogenic protein suggests that high EPO inhibits adipogenesis and osteogenesis. These multi-tissue responses underscore the pleiotropic potential of the EPO response and may contribute to various physiological manifestations accompanying anemia or ischemic response and pharmacological uses of EPO.

7.
Cell Biol Int ; 43(2): 192-206, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30571852

RESUMO

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 , Fosforilação/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único , Pirazóis/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Fatores de Transcrição STAT/metabolismo
8.
Vitam Horm ; 105: 101-120, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28629513

RESUMO

Erythropoietin (EPO), known primarily for its erythropoietic activity, is commonly used clinically to treat anemia of chronic kidney disease. However, the expression of EPO receptor (EpoR) beyond erythroid tissue provides for potential extrahematopoietic effects of EPO, including EPO regulation of metabolic homeostasis (Zhang et al., 2014). Small clinical studies have shown that EPO treatment in patients with end-stage renal disease improved glycemic control and insulin sensitivity. Studies in animal models have shown that EPO regulation of metabolism is mainly attributed to its response in fat, and the hypothalamus-pituitary axis (Dey et al., 2016; Dey, Scullen, & Noguchi, 2015; Teng, Gavrilova, et al., 2011; Wang et al., 2013) and is not dependent on its hematopoietic activity. EpoR expression in the hypothalamus is localized to the neurons expressing proopiomelanocortin (POMC) in the arcuate nucleus region, the most important site in the brain for the regulation of physiological energy expenditure. EPO treatment increases POMC production in anorexigenic POMC neurons in the hypothalamus. In the pituitary, EPO modulates the secretion of the POMC-derived peptide, adrenocorticotropic hormone (ACTH) that regulates physiological and metabolic stress response. With EPO produced by cells in the brain, such as astrocytes, and with EPO-stimulated POMC expression in the hypothalamus and EPO-inhibited ACTH secretion in the pituitary, EPO signaling contributes to the hypothalamic-pituitary axis as a major regulator of glucose metabolism and energy homeostasis.


Assuntos
Eritropoetina/metabolismo , Sistema Hipotálamo-Hipofisário/fisiologia , Animais , Eritropoetina/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Hipotálamo/fisiologia , Hipófise/fisiologia
9.
Mol Carcinog ; 56(2): 567-579, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27341002

RESUMO

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/análise
10.
Clin J Am Soc Nephrol ; 10(10): 1822-30, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26358266

RESUMO

BACKGROUND AND OBJECTIVES: Recombinant human erythropoietin (epoetin) is used routinely to increase blood hemoglobin levels in patients with ESRD and anemia. Although lower doses of epoetin are required to achieve equivalent hemoglobin responses when administered subcutaneously rather than intravenously, standard practice has been to administer epoetin to patients on hemodialysis intravenously. Randomized trials of alternative epoetin treatment regimens in patients with kidney failure have shown that risks of cardiovascular complications and death are related to the dose levels of epoetin used. Therefore, given the dose-sparing advantages of subcutaneous epoetin administration, the possibility that treatment of patients on hemodialysis with subcutaneous epoetin might be associated with more favorable outcomes compared with intravenous treatment was investigated. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A retrospective cohort study of 62,710 adult patients on hemodialysis treated with either intravenous or subcutaneous epoetin-α and enrolled in the Centers for Medicare and Medicaid Services ESRD Clinical Performance Measures Project from 1997 to 2005 was carried out. Risks of death and/or hospitalization for cardiovascular complications (adverse composite event outcomes) during 2 years of follow-up were determined in relationship to epoetin dose and route of administration (intravenous versus subcutaneous) by multivariate Cox proportional hazard modeling adjusted for demographics and clinical parameters. RESULTS: Epoetin doses used to achieve equivalent hemoglobin responses in study patients were, on average, 25% higher when epoetin was administered intravenously rather than subcutaneously (as expected). Moreover, adverse composite event outcomes were found to be significantly more likely to occur during follow-up for patients on hemodialysis managed with intravenous rather than subcutaneous epoetin (adjusted hazard ratio for adverse events within 1 year [intravenous versus subcutaneous] was 1.11 [95% confidence interval, 1.04 to 1.18]). CONCLUSIONS: This study finds that treatment of patients on hemodialysis with subcutaneous epoetin is associated with more favorable clinical outcomes than those associated with intravenous epoetin treatment.


Assuntos
Anemia/tratamento farmacológico , Doenças Cardiovasculares/epidemiologia , Epoetina alfa/administração & dosagem , Hematínicos/administração & dosagem , Falência Renal Crônica/mortalidade , Falência Renal Crônica/terapia , Administração Intravenosa , Idoso , Anemia/sangue , Doenças Cardiovasculares/etiologia , Epoetina alfa/efeitos adversos , Feminino , Insuficiência Cardíaca/epidemiologia , Insuficiência Cardíaca/etiologia , Hematínicos/efeitos adversos , Hemoglobinas/metabolismo , Hospitalização/estatística & dados numéricos , Humanos , Injeções Subcutâneas , Falência Renal Crônica/complicações , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/etiologia , Diálise Renal , Estudos Retrospectivos , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/etiologia , Estados Unidos/epidemiologia
11.
PLoS One ; 8(2): e55699, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383344

RESUMO

Nitric oxide (NO) appears to play an important role in the regulation of thrombosis and hemostasis by inhibiting platelet function. The discovery of NO generation by reduction of nitrite (NO2⁻) and nitrate (NO3⁻) in mammals has led to increased attention to these anions with respect to potential beneficial effects in cardiovascular diseases. We have previously shown that nitrite anions at 0.1 µM inhibit aggregation and activation of human platelet preparations in vitro in the presence of red blood cells and this effect was enhanced by deoxygenation, an effect likely due to NO generation. In the present study, we hypothesized that nitrite and nitrate derived from the diet could also alter platelet function upon their conversion to NO in vivo. To manipulate the levels of nitrite and nitrate in mouse blood, we used antibiotics, NOS inhibitors, low nitrite/nitrate (NOx) diets, endothelial NOS knock-out mice and also supplementation with high levels of nitrite or nitrate in the drinking water. We found that all of these perturbations affected nitrite and nitrate levels but that the lowest whole blood values were obtained by dietary restriction. Platelet aggregation and ATP release were measured in whole blood and the results show an inverse correlation between nitrite/nitrate levels and platelet activity in aggregation and ATP release. Furthermore, we demonstrated that nitrite-supplemented group has a prolonged bleeding time compared with control or low NOx diet group. These results show that diet restriction contributes greatly to blood nitrite and nitrate levels and that platelet reactivity can be significantly affected by these manipulations. Our study suggests that endogenous levels of nitrite and nitrate may be used as a biomarker for predicting platelet function and that dietary manipulation may affect thrombotic processes.


Assuntos
Plaquetas/fisiologia , Nitratos/sangue , Nitritos/sangue , Agregação Plaquetária/fisiologia , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Biomarcadores/sangue , Plaquetas/efeitos dos fármacos , Suplementos Nutricionais , Medições Luminescentes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitratos/farmacologia , Óxido Nítrico Sintase Tipo III/genética , Nitritos/farmacologia , Agregação Plaquetária/efeitos dos fármacos
12.
BMC Genomics ; 14: 116, 2013 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-23425329

RESUMO

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/metabolismo
13.
J Transl Med ; 10: 116, 2012 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-22676255

RESUMO

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.


Assuntos
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/metabolismo
14.
J Biomed Biotechnol ; 2011: 373781, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21541227

RESUMO

Erythropoietin (Epo) is a pleotropic cytokine with several nonhematopoietic tissue effects. High-dose Epo treatment-mediated effects on body weight, fat mass and glucose tolerance have recently been reported, thus extending its pleotropic effects to fat and glucose metabolism. However, the exact dose range of Epo treatment required for such effects remains unidentified to date. We investigated Epo dosage effect (up to 1000 U/kg) on hematocrit, body weight, body composition, glucose metabolism, food intake, and physical activity, during high-fat diet-induced obesity. We report that Epo doses (1000, 600, 300, and 150 U/kg) significantly reduced body weight gain and fat mass, while, only Epo doses of 300 U/kg and higher significantly affected glucose tolerance. None of the tested Epo doses showed any detectable effects on food intake, and only 1000 U/kg dose significantly increased physical activity, suggesting that these parameters may only be partially responsible for the metabolic effects of Epo treatment.


Assuntos
Dieta , Eritropoetina/administração & dosagem , Eritropoetina/uso terapêutico , Obesidade/tratamento farmacológico , Adiposidade/efeitos dos fármacos , Animais , Gorduras na Dieta , Relação Dose-Resposta a Droga , Epoetina alfa , Eritropoetina/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Glucose/metabolismo , Hematócrito , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Obesidade/induzido quimicamente , Proteínas Recombinantes , Ganho de Peso/efeitos dos fármacos
15.
Cytokine ; 54(2): 129-35, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21324713

RESUMO

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.


Assuntos
Endotélio Vascular/metabolismo , Eritropoetina/farmacologia , Hipóxia/metabolismo , Pulmão/irrigação sanguínea , Microvasos/metabolismo , Óxido Nítrico/metabolismo , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/enzimologia , Endotélio Vascular/patologia , Humanos , Hipóxia/patologia , Microvasos/enzimologia , Microvasos/patologia , Nitratos/metabolismo , Óxido Nítrico Sintase/metabolismo , Nitritos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
J Neurotrauma ; 27(1): 205-15, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19715391

RESUMO

Erythropoietin (EPO), essential for erythropoiesis, provides neuroprotection. The EPO receptor (EPOR) is expressed in both neural and non-neural cells in the brain. This study was designed to test the hypothesis that EPO provides beneficial therapeutic effects, even in the absence of the neural EPOR. In this study, EPOR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue, but not in the neural cells. Anesthetized young adult female EPOR-null and wild-type mice were subjected to traumatic brain injury (TBI) induced by controlled cortical impact. EPO (5000 U/kg) or saline was intraperitoneally administered at 6 h and 3 and 7 days post-injury. Sensorimotor and spatial learning functions were assessed. Expression of EPOR and its downstream signal proteins were evaluated by Western blot analysis. Our data demonstrated that EPO treatment significantly reduced cortical tissue damage and hippocampal cell loss, and improved spatial learning following TBI in both the wild-type and EPOR-null mice. EPO treatment significantly improved sensorimotor functional recovery, with better outcomes in the wild-type mice. EPO treatment upregulated anti-apoptotic proteins (p-Akt and Bcl-XL) in the ipsilateral hippocampus and cortex of the injured wild-type and EPOR-null mice. These data demonstrate that EPO significantly provides neuroprotection following TBI, even in the absence of EPOR in the neural cells, suggesting that its therapeutic benefits may be mediated through vascular protection.


Assuntos
Lesões Encefálicas/tratamento farmacológico , Eritropoetina/farmacologia , Fármacos Neuroprotetores/farmacologia , Receptores da Eritropoetina/genética , Animais , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/fisiopatologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Eritropoetina/metabolismo , Eritropoetina/uso terapêutico , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/etiologia , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Degeneração Neural/tratamento farmacológico , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Fármacos Neuroprotetores/uso terapêutico , Regiões Promotoras Genéticas/genética , Receptores da Eritropoetina/metabolismo , Resultado do Tratamento
17.
Exp Hematol ; 37(10): 1230-7, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19576950

RESUMO

OBJECTIVE: We 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. MATERIALS AND METHODS: Using 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. RESULTS: Hydroxyurea (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). CONCLUSION: These 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.


Assuntos
Células Endoteliais/efeitos dos fármacos , Células Eritroides/metabolismo , Macrófagos/efeitos dos fármacos , Óxido Nítrico/fisiologia , Células Estromais/fisiologia , gama-Globinas/biossíntese , Adulto , Animais , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Técnicas de Cocultura , Sinergismo Farmacológico , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Comunicação Parácrina/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Estromais/efeitos dos fármacos , gama-Globinas/genética
18.
Brain Res ; 1230: 247-57, 2008 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-18657521

RESUMO

Erythropoietin (EPO) and its receptor (EPOR), essential for erythropoiesis, are expressed in the nervous system. Recombinant human EPO treatment promotes functional outcome after traumatic brain injury (TBI) and stroke, suggesting that the endogenous EPO/EPOR system plays an important role in neuroprotection and neurorestoration. This study was designed to investigate effects of the EPOR on histological and functional outcomes after TBI. Experimental TBI was induced in adult EPOR-null and wild-type mice by controlled cortical impact. Neurological function was assessed using the modified Morris Water Maze and footfault tests. Animals were sacrificed 35 days after injury and brain sections stained for immunohistochemistry. As compared to the wild-type injured mice, EPOR-null mice did not exhibit higher susceptibility to TBI as exemplified by tissue loss in the cortex, cell loss in the dentate gyrus, impaired spatial learning, angiogenesis and cell proliferation. We observed that less cortical neurogenesis occurred and that sensorimotor function (i.e., footfault) was more impaired in the EPOR-null mice after TBI. Co-accumulation of amyloid precursor protein (axonal injury marker) and calcium was observed in the ipsilateral thalamus in both EPOR-null and wild-type mice after TBI with more calcium deposits present in the wild-type mice. This study demonstrates for the first time that EPOR null in the nervous system aggravates sensorimotor deficits, impairs cortical neurogenesis and reduces thalamic calcium precipitation after TBI.


Assuntos
Lesões Encefálicas/patologia , Sistema Nervoso Central/fisiologia , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/fisiologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Antimetabólitos , Lesões Encefálicas/metabolismo , Bromodesoxiuridina , Cálcio/metabolismo , Contagem de Células , Proliferação de Células , Sistema Nervoso Central/metabolismo , Córtex Cerebral/patologia , Giro Denteado/patologia , Feminino , Imunofluorescência , Hipocampo/patologia , Imuno-Histoquímica , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Neurônios/fisiologia , Desempenho Psicomotor/fisiologia
19.
Blood ; 111(3): 1117-23, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17993617

RESUMO

Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We have shown that nitric oxide (NO) and the soluble guanylyl cyclase (sGC) pathways are involved in hydroxyurea induction of HbF levels in erythroid progenitor cells (EPCs). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cyclic adenosine monophosphate (cAMP) levels, but guanosine 3',5'-cyclic monophosphate (cGMP) levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in EPCs. The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 pM cGMP/minute/ng of purified sGC, similar to induction by 1 muM DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPCs. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF.


Assuntos
Células Eritroides/efeitos dos fármacos , Células Eritroides/enzimologia , Guanilato Ciclase/metabolismo , Hidroxiureia/farmacologia , Ácido Nitroso/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Eritroides/citologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Nucleotídeos Cíclicos/metabolismo , Guanilil Ciclase Solúvel
20.
Exp Hematol ; 35(8): 1209-18, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17662889

RESUMO

OBJECTIVE: Sickle cell anemia is a genetic blood disease resulting from production of mutant beta-globin (beta(S)) and has severe clinical consequences. It is known that a higher cellular gamma-globin level, e.g., higher ratio of cellular gamma-globin to beta(S)-globin (gamma/beta(S) ratio), inhibits sickle hemoglobin (HbS) polymerization tendency. Hence, therapeutic treatment of sickle cell anemia has been focused on introducing gamma-globin gene into red blood cells to increase the cellular gamma/beta(S) ratio. Here, we have introduced ribozymes and small interfering RNAs (siRNAs) against beta(S)-globin mRNA into blood cells as a means to increase the gamma/beta(S) ratio. MATERIALS AND METHODS: Single and multiribozymes against beta(S)-globin mRNA have been tested in vitro and in human erythroleukemia K562beta(S) cells that stably express exogenous beta(S)-globin gene. Primary human hematopoietic progenitor cells were also transfected with multiribozyme and the gamma/(gamma + beta) ratio determined and compared with cells transfected with long hairpin beta-globin cDNA and synthetic siRNA genes. RESULTS: We have found that the multiribozyme zb21A containing two ribozyme units effectively reduces beta(S)-globin mRNA both in vitro and in K562beta(S) cells. The gamma-globin mRNA to beta(S)-globin mRNA ratio in the multiribozyme transfected cells is about a factor of 2 more than that in the control cells. We have also found that the gamma/(gamma + beta) ratio in the transfected hematopoietic progenitor cells is increased by more than twofold in cells treated with multiribozyme zb21A or siRNA ib5. CONCLUSION: Our results suggest that introducing multiribozymes or siRNAs into red blood cells is comparable in their effectiveness to increase the ratio of cellular gamma-globin mRNA to beta- or beta(S)-globin mRNA, providing possible strategies to increase the effectiveness of gamma-globin gene transfer as gene therapy for treatment of patients with sickle cell anemia.


Assuntos
Regulação da Expressão Gênica , Globinas/genética , RNA Mensageiro/genética , Anemia Falciforme/genética , Sequência de Bases , Linhagem Celular Tumoral , Éxons , Globinas/fisiologia , Humanos , Células K562 , Dados de Sequência Molecular , Plasmídeos , RNA Interferente Pequeno/genética
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