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2.
J Biol Chem ; 289(16): 11512-11521, 2014 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-24616094

RESUMO

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4ß1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.


Assuntos
Anemia Falciforme/metabolismo , Antidrepanocíticos/farmacologia , Moléculas de Adesão Celular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/biossíntese , Células Endoteliais/metabolismo , Hidroxiureia/farmacologia , Sistema do Grupo Sanguíneo Lutheran/metabolismo , Reticulócitos/metabolismo , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Anemia Falciforme/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Moléculas de Adesão Celular/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Células Endoteliais/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Células K562 , Sistema do Grupo Sanguíneo Lutheran/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Reticulócitos/patologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
3.
Haematologica ; 98(6): 862-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23403312

RESUMO

Microparticles are cell membrane-derived microvesicles released during cell apoptosis and activation processes. They have been described as bio-markers in various vascular diseases, including sickle cell anemia, and associated with an increased risk of thrombosis. We investigated the effects of fetal hemoglobin level, a factor known to modulate the clinical expression of sickle cell anemia, and that of hydroxycarbamide treatment which reduces the frequency of vasoocclusive crises, the canonical clinical manifestation of the disease, on both the plasma concentration and the cellular origin of circulating microparticles. Flow cytometry was used to characterize microparticles in 62 sickle cell anemia children at steady state aged 2 months-16 years; 13 of them were treated with hydroxycarbamide. In untreated children, we observed negative correlations between fetal hemoglobin levels and the absolute plasma concentration of microparticles as well as that of microparticles specifically derived from platelets, erythrocytes, and monocytes. Compared to untreated children, those treated with hydroxyurea showed lower concentrations of total microparticles as a consequence of decreased microparticles shed by platelets and erythrocytes. In conclusion, in our sickle cell patients, neonatal decline of fetal hemoglobin coincided with an increase in circulating microparticles derived from erythrocytes, platelets, and monocytes. Hydroxyurea treatment was associated with a decrease in microparticles derived from erythrocytes and platelets.


Assuntos
Anemia Falciforme/metabolismo , Micropartículas Derivadas de Células/metabolismo , Hemoglobina Fetal/metabolismo , Hidroxiureia/farmacologia , Adolescente , Anemia Falciforme/tratamento farmacológico , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Criança , Pré-Escolar , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Hidroxiureia/uso terapêutico , Lactente , Masculino , Monócitos/efeitos dos fármacos , Monócitos/metabolismo
4.
Am J Respir Crit Care Med ; 184(12): 1358-66, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21885626

RESUMO

RATIONALE: Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation of unknown pathogenesis. OBJECTIVES: To investigate whether telomere dysfunction and senescence of pulmonary vascular endothelial cells (P-ECs) induce inflammation in COPD. METHODS: Prospective comparison of patients with COPD and age- and sex-matched control smokers. Investigation of mice null for telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. MEASUREMENTS AND MAIN RESULTS: In situ lung specimen studies showed a higher percentage of senescent P-ECs stained for p16 and p21 in patients with COPD than in control subjects. Cultured P-ECs from patients with COPD exhibited early replicative senescence, with decreased cell-population doublings, a higher percentage of ß-galactosidase-positive cells, reduced telomerase activity, shorter telomeres, and higher p16 and p21 mRNA levels at an early cell passage compared with control subjects. Senescent P-ECs released cytokines and mediators: the levels of IL-6, IL-8, monocyte chemotactic protein (MCP)-1, Hu-GRO, and soluble intercellular adhesion molecule (sICAM)-1 were elevated in the media of P-ECs from patients compared with control subjects at an early cell passage, in proportion to the senescent P-EC increase and telomere shortening. Up-regulation of MCP-1 and sICAM-1 led to increased monocyte adherence and migration. The elevated MCP-1, IL-8, Hu-GROα, and ICAM-1 levels measured in lungs from patients compared with control subjects correlated with P-EC senescence criteria and telomere length. In Tert(-/-) and/or Terc(-/-) mouse lungs, levels of the corresponding cytokines (MCP-1, IL-8, Hu-GROα, and ICAM-1) were also altered, despite the absence of external stimuli and in proportion to telomere dysfunction. CONCLUSIONS: Telomere dysfunction and premature P-EC senescence are major processes perpetuating lung inflammation in COPD.


Assuntos
Endotélio Vascular/ultraestrutura , Inflamação/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Encurtamento do Telômero , Adulto , Animais , Estudos de Casos e Controles , Feminino , Humanos , Análise dos Mínimos Quadrados , Masculino , Análise por Pareamento , Camundongos , Camundongos Knockout , Estudos Prospectivos , Fumar/efeitos adversos
5.
Clin Hemorheol Microcirc ; 47(1): 15-25, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21321404

RESUMO

Strenuous exercise is associated with an inflammatory response involving the activation of several types of blood cells. In order to document the specific activation of these cell types, we studied the effect of three maximal exercise tests conducted to exhaustion on the quantitative and qualitative pattern of circulating cell-derived microparticles and inflammatory molecules in healthy subjects. This study mainly indicated that the plasma concentration of microparticles from platelets and polymorphonuclear neutrophils (PMN) was increased immediately after the strenuous exercise. In addition, the increase in plasma concentration of microparticles from PMN and platelets was still observed after 2 hours of recovery. A similar pattern was observed for the IL-6 plasma level. In contrast, no change was observed for either soluble selectins or plasma concentration of microparticles from red blood cells, monocytes and endothelial cells. In agreement, sVCAM-1 and sICAM-1 levels were not changed by the exercise. We conclude that a strenuous exercise is accompanied by platelet- and PMN-derived microparticle production that probably reflects the activation of these two cell types.


Assuntos
Plaquetas/citologia , Micropartículas Derivadas de Células/metabolismo , Exercício Físico , Neutrófilos/citologia , Adulto , Humanos , Interleucina-6/sangue , Masculino , Adulto Jovem
6.
J Appl Physiol (1985) ; 110(5): 1196-203, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21252221

RESUMO

Chronic obstructive pulmonary disease (COPD) is a major risk factor for cardiovascular disease. Polycythemia, a common complication of hypoxic COPD, may affect systemic vascular function by altering blood viscosity, vessel wall shear stress (WSS), and endothelium-derived nitric oxide (NO) release. Here, we evaluated the effects of hypoxia-related polycythemia on systemic endothelial function in patients with COPD. We investigated blood viscosity, WSS, and endothelial function in 15 polycythemic and 13 normocythemic patients with COPD of equal severity, by recording brachial artery diameter variations in response to hyperemia and by using venous occlusion plethysmography (VOP) to measure forearm blood flow (FBF) responses to a brachial artery infusion of acetylcholine (ACh), bradykinin (BK), sodium nitroprusside (SNP), substance P (SP), isoptin, and N-monomethyl-L-arginine (L-NMMA). At baseline, polycythemic patients had higher blood viscosity and larger brachial artery diameter than normocythemic patients but similar calculated WSS. Flow-mediated brachial artery vasodilation was increased in the polycythemic patients, in proportion to the hemoglobin levels. ACh-induced vasodilation was markedly impaired in the polycythemic patients and negatively correlated with hemoglobin levels. FBF responses to endothelium- (BK, SP) and non-endothelium-dependent (SNP, isoptin) vasodilators were not significantly different between the two groups. L-NMMA infusion induced a similar vasoconstrictor response in both groups, in accordance with their similar baseline WSS. In conclusion, systemic arteries in polycythemic patients adjust appropriately to chronic or acute WSS elevations by appropriate basal and stimulated NO release. Overall, our results suggest that moderate polycythemia has no adverse effect on vascular function in COPD.


Assuntos
Endotélio Vascular/fisiopatologia , Hipóxia/complicações , Hipóxia/fisiopatologia , Policitemia/etiologia , Policitemia/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Velocidade do Fluxo Sanguíneo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
7.
Blood ; 116(12): 2152-9, 2010 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-20566895

RESUMO

Sickle cell disease is characterized by painful vaso-occlusive crises during which abnormal interactions between erythroid adhesion molecules and vessel-wall proteins are thought to play a critical role. Hydroxyurea, the only drug with proven benefit in sickle cell disease, diminishes these interactions, but its mechanism of action is not fully understood. We report that, under hydroxyurea, expression of the unique erythroid laminin receptor Lu/BCAM was increased, but red blood cell adhesion to laminin decreased. Because Lu/BCAM phosphorylation is known to activate cell adhesion to laminin, it was evaluated and found to be dramatically lower in hydroxyurea-treated patients. Analysis of the protein kinase A pathway showed decreased intracellular levels of the upstream effector cyclic adenosine monophosphate during hydroxyurea treatment. Using a cellular model expressing recombinant Lu/BCAM, we showed that hydroxyurea led to decreased intracellular cyclic adenosine monophosphate levels and diminished Lu/BCAM phosphorylation and cell adhesion. We provide evidence that hydroxyurea could reduce abnormal sickle red blood cell adhesion to the vascular wall by regulating the activation state of adhesion molecules independently of their expression level.


Assuntos
Anemia Falciforme/sangue , Moléculas de Adesão Celular/metabolismo , Adesão Celular/efeitos dos fármacos , Eritrócitos Anormais/patologia , Hidroxiureia/farmacologia , Sistema do Grupo Sanguíneo Lutheran/metabolismo , AMP Cíclico/análise , Humanos , Células K562 , Laminina/metabolismo , Fosforilação/efeitos dos fármacos
8.
Haematologica ; 95(11): 1841-8, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20562314

RESUMO

BACKGROUND: Abnormal interactions between red blood cells, leukocytes and endothelial cells play a critical role in the occurrence of the painful vaso-occlusive crises associated with sickle cell disease. We investigated the interaction between circulating leukocytes and red blood cells which could lead to aggregate formation, enhancing the incidence of vaso-occlusive crises. DESIGN AND METHODS: Blood samples from patients with sickle cell disease (n=25) and healthy subjects (n=5) were analyzed by imaging and classical flow cytometry after density gradient separation. The identity of the cells in the peripheral blood mononuclear cell layer was determined using antibodies directed specifically against white (anti-CD45) or red (anti-glycophorin A) blood cells. RESULTS: Aggregates between red blood cells and peripheral blood mononuclear cells were visualized in whole blood from patients with sickle cell disease. The aggregation rate was 10-fold higher in these patients than in control subjects. Both mature red blood cells and reticulocytes were involved in these aggregates through their interaction with mononuclear cells, mainly with monocytes. The size of the aggregates was variable, with one mononuclear cell binding to one, two or several red blood cells. Erythroid Lu/basal cell adhesion molecule and α(4)ß(1) integrin were involved in aggregate formation. The aggregation rate was lower in patients treated with hydroxycarbamide than in untreated patients. CONCLUSIONS: Our study gives visual evidence of the existence of circulating red blood cell-peripheral blood mononuclear cell aggregates in patients with sickle cell disease and shows that these aggregates are decreased during hydroxycarbamide treatment. Our results strongly suggest that erythroid Lu/basal cell adhesion molecule proteins are implicated in these aggregates through their interaction with α(4)ß(1) integrin on peripheral blood mononuclear cells.


Assuntos
Anemia Falciforme/metabolismo , Moléculas de Adesão Celular/metabolismo , Eritrócitos/metabolismo , Integrina alfa4beta1/metabolismo , Sistema do Grupo Sanguíneo Lutheran/metabolismo , Monócitos/metabolismo , Adulto , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Anemia Falciforme/patologia , Antidrepanocíticos/administração & dosagem , Agregação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Eritrócitos/patologia , Feminino , Humanos , Hidroxiureia/administração & dosagem , Masculino , Monócitos/patologia
9.
Biochemistry ; 47(25): 6519-30, 2008 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-18512959

RESUMO

Thymine-DNA glycosylases (TDGs) initiate base excision repair by debasification of the erroneous thymine or uracil nucleotide in G.T and G.U mispairs which arise at high frequency through spontaneous or enzymatic deamination of methylcytosine and cytosine, respectively. Human TDG has furthermore been shown to have a functional role in transcription and epigenetic regulation through the interaction with transcription factors from the nuclear receptor superfamily, transcriptional coregulators, and a DNA methyltransferase. The TDG N-terminus encodes regulatory functions, as it assures both G.T versus G.U specificity and contains the sites for interaction and posttranslational modification by transcription-related activities. While the molecular function of the evolutionarily conserved central catalytic domain of TDG in base excision repair has been elucidated by determination of its three-dimensional structure, the mechanisms by which the N-terminus exerts its regulatory roles, as well as the function of TDG in transcription regulation, remain to be understood. We describe here the residual structure of the TDG N-terminus in both contexts of the isolated domain and the entire protein. These studies lead to the characterization of a small structural domain in the TDG N-terminal region preceding the catalytic core and coinciding with the region of functional regulation of TDG's activities. This regulatory domain exhibits a small degree of organization and is implicated in dynamic molecular interactions with the catalytic domain and nonselective interactions with double-stranded DNA, providing a molecular explanation for the evolutionarily acquired G.T mismatch processing activity of TDG.


Assuntos
Domínio Catalítico , Reparo do DNA , Timina DNA Glicosilase/química , Timina DNA Glicosilase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , DNA/química , DNA/genética , DNA/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Prolina/química , Prolina/genética , Prolina/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Timina DNA Glicosilase/genética
10.
Haematologica ; 91(9): 1277-8, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16956834

RESUMO

The association of endothelin 1 (ET-1) and endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphisms (G5665T and T8002C, VNTR and T-786C respectively) with the occurrence of acute chest syndrome and painful vaso-occlusive crises was evaluated in homozygous SS children. This retrospective study reveals that ET-1 T8002 and ecNOS C-786 alleles are associated with, respectively, an increased and a decreased risk of acute chest syndrome.


Assuntos
Anemia Falciforme/complicações , Anemia Falciforme/genética , Endotelina-1/genética , Óxido Nítrico Sintase Tipo III/genética , Adolescente , Dor no Peito/genética , Criança , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Dor , Polimorfismo Genético , Doenças Vasculares
11.
Am J Hematol ; 81(5): 377-9, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16628735

RESUMO

Enhanced erythrocyte destruction in sickle cell anemia results in chronic hyperbilirubinemia. Only a subset of patients develop cholelithiasis. UGT1A1 promoter polymorphism is associated both with unconjugated bilirubin level and elevated risk for cholelithiasis in such subset. Here, we investigated the role of alpha-thalassemia, yet another genetic factor that modulates hemolysis, in conferring protection from cholelithiasis. We show that, although alpha-thalassemia is associated with modest reduction in hemolysis and unconjugated bilirubin level, UGT1A1 polymorphism outweighs its effect on cholethiogenesis in sickle cell anemia patients.


Assuntos
Anemia Falciforme/genética , Colelitíase/genética , Deleção de Genes , Glucuronosiltransferase/genética , Polimorfismo Genético , Talassemia alfa/genética , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/complicações , Bilirrubina/sangue , Criança , Colelitíase/sangue , Colelitíase/etiologia , Globinas/genética , Humanos , Talassemia alfa/sangue , Talassemia alfa/complicações
12.
Haematologica ; 90(2): 188-99, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15710570

RESUMO

BACKGROUND AND OBJECTIVES: High levels of erythrocyte destruction in sickle cell anemia (SCA) result in chronic hyperbilirubinemia, with cholelithiasis occurring in a subset of patients. We investigated whether susceptibility to cholelithiasis in SCA was associated with the promoter polymorphism of the 5?-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) gene encoding a key enzyme in bilirubin catabolism. DESIGN AND METHODS: We determined the frequencies of UGT1A1 promoter alleles in 171 SCA children and 153 SCA adults regularly followed for a number of years at the Guadeloupe sickle cell center. These patients had undergone liver/biliary tree ultrasound scans every year. We analyzed the relationships between the various UGT1A1 promoter alleles and hemoglobin levels, steady-state total and unconjugated bilirubin concentrations and the frequency of cholelithiasis. RESULTS: In both children and adults, (TA)6 was less frequent and (TA)7 more frequent in patients with cholelithiasis than in those without this condition. Total and unconjugated bilirubin levels and the frequency of cholelithiasis were significantly higher in patients with (TA)7/(TA)7 and (TA)7/(TA)8 genotypes than in those with other genotypes. Those homozygous for (TA)6 or carrying at least one (TA)5 allele had the lowest total and unconjugated bilirubin levels and were least likely to have cholelithiasis. Patients with (TA)6/(TA)7 and (TA)6/(TA)8 genotypes presented intermediate values. Kaplan-Meier analysis of cholelithiasis-free survival in children demonstrated an early age-at-onset for cholelithiasis in patients with (TA)7/(TA)7 and (TA)7/(TA)8 genotypes. INTERPRETATIONS AND CONCLUSIONS: This study shows that the UGT1A1 gene promoter polymorphism is a major genetic risk factor modifying the frequency and age-at-onset of cholelithiasis in SCA patients.


Assuntos
Anemia Falciforme/complicações , Anemia Falciforme/genética , Colelitíase/complicações , Colelitíase/genética , Glucuronosiltransferase/genética , Polimorfismo Genético , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Feminino , Genótipo , Hemoglobinas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Fatores de Risco
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