RESUMEN
Advanced glycation end products (AGE) result from a chemical reaction between the carbonyl group of reducing sugar and the nucleophilic NH2 of a free amino acid or a protein; lysine and arginine being the main reactive amino acids on proteins. Following this first step, a molecular rearrangement occurs, rearrangement of Amadori resulting to the formation of Maillard products. Glycation can cause the clouding of the lens by inducing reactions crosslinking proteins. Specialized receptors (RAGE, Galectin 3 ) bind AGE. The binding to the receptor causes the formation of free radicals, which have a deleterious effect because they are powerful oxidizing agents, but also play the role of intracellular messenger, altering the cell functions. This is especially true at the level of endothelial cells: the attachment of AGE to RAGE receptor causes an increase in vascular permeability. AGE binding to endothelium RAGE and to monocytes-macrophages, led to the production of cytokines, growth factors, to the expression of adhesion molecules, and the production of procoagulant activity. Diabetic retinopathy is related to excessive secretion of vascular growth factor (vascular endothelial growth factor [VEGF]). AGE-RAGE receptor binding causes the synthesis and secretion of VEGF. Increased permeability, facilitation of leukocyte migration, the production of reactive oxygen species, cytokines and VEGF suggest that the AGE could be an element of a cascade of reactions responsible for the diabetic angiopathy and vascular damages observed during aging and chronic renal failure. Balanced diet or some drugs can limit the deleterious effect of AGE.
Asunto(s)
Productos Finales de Glicación Avanzada/metabolismo , Productos Finales de Glicación Avanzada/genética , Productos Finales de Glicación Avanzada/fisiología , Humanos , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Factores de RiesgoRESUMEN
BACKGROUND: Retinal vein occlusion (RVO) is a common cause of permanent loss of vision. The pathophysiology is uncertain, although enhanced erythrocyte aggregation and blood hyperviscosity have been observed. Increased red blood cell (RBC) adhesion has been associated with vascular complications in several diseases, such as sickle cell anemia, diabetes mellitus or polycythemia vera. OBJECTIVES: To measure RBC adhesion to endothelial cells in RVO and to explore the molecular basis of the adhesion process. PATIENTS AND METHODS: We assessed RBC adhesion to endothelial cells and adhesion molecule expression among 32 patients with RVO. Patients with disease known to alter RBC adhesion were excluded (n = 8), and further investigation was conducted in 20 patients with central retinal vein occlusion (CRVO) and four patients with retinal artery occlusion (RAO), compared with 25 normal subjects. RESULTS: Under static conditions, adhesion of CRVO RBC was increased (135 ± 7 × 10(2) mm(-2)) compared with RAO RBC (63 ± 5 × 10(2) mm(-2)) (P < 0.01) and normal control RBC (37 ± 3 × 10(2) mm(-2)) (P < 0.001). Under flow conditions, CRVO RBC adhered in greater numbers than normal RBC (P < 0.001). Phosphatidylserine (PS) expression on CRVO RBC was 2.4-fold higher than controls and correlated with RBC adhesion (P = 0.001). In static conditions, specific antibodies against PS receptor and annexin V inhibited RBC adhesion. In flow conditions, the inhibitory effect was in the same range with antibodies but was 2-fold higher with annexin V. CONCLUSION: Increased CRVO RBC adhesion is mediated by PS RBC and endothelial PS receptor. This phenomenon may be one of the factors responsible for CRVO.
Asunto(s)
Eritrocitos/citología , Fosfatidilserinas/sangre , Oclusión de la Vena Retiniana/sangre , Vasos Retinianos/metabolismo , Adulto , Anciano , Anexina A5/química , Adhesión Celular , Agregación Eritrocitaria , Femenino , Hematócrito , Humanos , Masculino , Persona de Mediana Edad , Fosfatidilserinas/química , Arteria Retiniana/citologíaRESUMEN
The extent of red blood cell adhesion is correlated with the incidence of vascular complications and the severity of the disease. Patients with sickle cell anemia (HbSS) experience vasoocclusive episodes. The adhesion of RBCs from HbSS patients is increased and related to VLA-4 exposure, which binds to vascular cell adhesion molecule (VCAM-1). Inter Cellular Adhesion Molecule (ICAM-1), CD31, CD36 and glycans are potential receptors for PfEMP1 of RBCs parasited by plasmodium falciparum. The incidence of vascular complications is very high in patients with diabetes mellitus. RBC adhesion is increased and statistically correlated with the severity of the angiopathy. Glycation of RBC membrane proteins is responsible for binding to the receptor for advanced glycation end products (RAGE). Polycythemia Vera (PV) is the most frequent myeloproliferative disorder and characterized by a high occurrence of thrombosis of mesenteric and cerebral vessels. PV is due to a mutation of the Janus kinase 2 (JAK2 V617F). This mutation stimulates erythropoiesis and is the cause of Lu/BCAM (CD239) phosphorylation, which potentiated the interaction with laminin alpha 5. The couple laminin alpha 5 endothelial and phosphorylated Lu/BCAM explained the increased adhesion of RBCs from patients PV to endothelium.
Asunto(s)
Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Eritrocitos/fisiología , Anemia de Células Falciformes/sangre , Animales , Diabetes Mellitus/sangre , Humanos , Malaria Falciparum/sangre , Policitemia Vera/sangre , Enfermedades Vasculares/etiologíaRESUMEN
AIM: Binding of advanced glycation end-products (AGEs) to the receptor for AGEs (RAGE) contributes to diabetic vascular complications. RAGE transcript splicing generates membrane-bound proteins [full-length (FL) and N-truncated (Nt)] and a soluble protein [endogenous secretory (esRAGE)] that may act as a decoy. We tested the effect of AGE-ligands on the regulation of RAGE isoforms and the consequences on red blood cell (RBC) adhesion. METHODS: RAGE isoforms were measured by real-time RT-PCR assay, using a LightCycler System, in human umbilical vein endothelial cells (HUVECs), incubated with either characterized AGEs [Nvarepsilon-(carboxymethyl)lysine human serum albumin (CML-HSA) and methylglyoxal-modified HSA (MG-HSA)] or with RBCs from diabetic patients (DRBCs). Inhibition of RAGE access was achieved by using blocking either anti-RAGE antibodies or recombinant RAGE. Adhesion of DRBCs to endothelium was measured under flow conditions using HUVECs stimulated with MG-HSA or CML-HSA. Antibodies directed to RBC membrane proteins were tested for blocking DRBC adhesion in static conditions. RESULTS: MG-HSA stimulated the expression of membrane-bound RAGE (FL+Nt) and esRAGE transcripts to similar extents, while CML-HSA and DRBC more selectively induced mRNA for FL and Nt-RAGE. Anti-RAGE antibody inhibited the effect of glycated proteins. Stimulation of HUVECs with CML-HSA enhanced DRBC adhesion, while MG-HSA had no effect. CD233 (band 3) was glycated in DRBC membrane, and anti-CD233 antibodies inhibited the adhesion of DRBCs, as did the anti-RAGE and anti-AGE antibodies. CONCLUSIONS: Receptor engagement by distinct AGEs differentially enhances expression of RAGE isoforms and DRBC adhesion. The CML-adduct, by facilitating adhesion, has more deleterious effects than MG-derived AGEs.
Asunto(s)
Eritrocitos/metabolismo , Regulación de la Expresión Génica , Productos Finales de Glicación Avanzada/metabolismo , Receptores Inmunológicos/metabolismo , Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Secuencia de Bases , Unión Competitiva , Adhesión Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/fisiopatología , Angiopatías Diabéticas/prevención & control , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Eritrocitos/química , Eritrocitos/fisiología , Hemoglobina Glucada/análisis , Productos Finales de Glicación Avanzada/síntesis química , Productos Finales de Glicación Avanzada/química , Humanos , Ligandos , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Alineación de Secuencia , Albúmina Sérica/química , Albúmina Sérica/metabolismoRESUMEN
Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Productos Finales de Glicación Avanzada/farmacología , Metaloproteinasa 2 de la Matriz/genética , Células Mesangiales/efectos de los fármacos , Anticuerpos/farmacología , Northern Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Proteínas de la Matriz Extracelular/metabolismo , Flavonoides/farmacología , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Humanos , Lisina/análogos & derivados , Lisina/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Células Mesangiales/citología , Células Mesangiales/metabolismo , Norleucina/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismo , alfa-Macroglobulinas/genética , alfa-Macroglobulinas/metabolismoRESUMEN
Recent studies shed new lights on the biological function of blood group antigens, such as the adhesion properties of the Lutheran (Lu) blood group antigens carried by the Lu/BCAM glycoproteins. The Lu/BCAM adhesion glycoproteins were first identified as laminin-10/11 erythroid receptors involved in RBC adhesion to endothelium in sickle cell anemia. Lu/BCAM mediated cell adhesion to laminin is stimulated by epinephrine, a physiological stress mediator, and is dependent of phosphorylation by protein kinase A. More recently, we demonstrated that constitutive phosphorylation of Lu/BCAM is also involved in abnormal RBC adhesion to endothelium in patients with polycythemia vera (PV), a frequent myeloproliferative disorders associated with the V617F mutation of the tyrosine kinase JAK2 leading to continuous stimulation of erythropoiesis. This observation suggests that Lu/BCAM could participate to the high incidence of vascular thrombosis that also characterizes PV disease. In mice, which do not express Lu/BCAM in erytroid tissues, invalidation of the Lu/BCAM gene provided evidence that Lu/BCAM gps, as laminin-alpha5 receptors, are involved in vivo in the maintenance of normal basement membrane organization in different non erythroid tissues since up to 90% of the mutant kidney glomeruli exhibited a reduced number of visible capillary lumens and irregular thickening of the glomerular basement membrane, while intestine exhibited smooth muscle coat thickening and disorganization. All these results further illustrate that minor blood group antigens might have important role under physiological and physiopathological conditions in erythroid and non erythroid tissues as well.
Asunto(s)
Anemia de Células Falciformes/sangre , Eritrocitos/patología , Sistema del Grupo Sanguíneo Lutheran/genética , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/fisiopatología , Animales , Adhesión Celular , Colforsina/farmacología , Humanos , Intestinos/patología , Riñón/patología , Glomérulos Renales/fisiología , Túbulos Renales/fisiología , Sistema del Grupo Sanguíneo Lutheran/metabolismo , Ratones , Ratones Noqueados , FosforilaciónRESUMEN
AIMS: The receptor for advanced glycation end-products (RAGE) has been implicated in diabetic microvascular complications, but several lines of evidence suggest that the soluble isoform of RAGE (sRAGE) may protect against AGE-mediated vessel damage. The characterized AGE Nepsilon-carboxymethyllysine (CML) is associated with diabetic microvascular complications. In the present study, we measured blood levels of sRAGE and CML-protein in diabetic patients with and without microvascular complications. METHODS: Thirty patients with type-2 diabetes were recruited into the study, comprising 20 who had no microvascular complications, and 10 who had both retinal and renal complications. sRAGE was measured in serum by ELISA, and CML by competitive ELISA. RESULTS: sRAGE blood levels were similar in both the controls and diabetic patients without microvascular complications. In patients with complications, the mean sRAGE blood level was significantly decreased (1068+/-231pg/mL) compared with diabetic patients without complications (P=0.028). CML-protein was increased in all diabetic patients, but to a higher extent in those who had microvascular complications. CONCLUSION: The association of low sRAGE with high CML-protein levels in diabetic patients who developed severe diabetic complications supports the hypothesis that sRAGE protects vessels against AGE-mediated diabetic microvascular damage.
Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/fisiopatología , Retinopatía Diabética/fisiopatología , Receptores Inmunológicos/sangre , Diabetes Mellitus Tipo 2/sangre , Nefropatías Diabéticas/sangre , Retinopatía Diabética/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lisina/análogos & derivados , Lisina/sangre , Masculino , Persona de Mediana Edad , Receptor para Productos Finales de Glicación AvanzadaRESUMEN
Lutheran (Lu) blood group and Basal Cell Adhesion Molecule (BCAM) antigens are both carried by two glycoprotein (gp) isoforms of the immunoglobulin superfamily representing receptors for laminin alpha5 chain. They are expressed in red blood cells, in endothelial cells of vascular capillaries and in epithelial cells of several tissues. Lu/BCAM gps are overexpressed in sickle red blood cells (SS RBCs). Stimulation of SS RBCs by epinephrine activates the PKA depending signaling pathway and induces reinforced Lu/BCAM-mediated adhesion to laminin10/11. We have analyzed the phosphorylation state of Lu/BCAM long isoform cytoplasmic tail and showed that it is phosphorylated by CKII, GSK3b and PKA. Phosphorylation of this isoform in transfected K562 cells is stimulated by effectors of the PKA pathway and induces cell adhesion to laminin10/11. Lu/BCAM gps are highly expressed in endothelial cells and exhibit potential integrin binding motifs. We showed that they interact with integrin alpha4beta1, the unique integrin expressed on the surface of young reticulocytes. Adhesion assays under flow conditions showed that SS RBCs adhere to primary human endothelial cells (HUVEC) after selective activation of intergin alpha4beta1 and that this adhesion is mediated by endothelial Lu/BCAM gps. Our studies show that Lu/BCAM gps expressed either on erythroid or on endothelial cells are involved in SS RBC-endothelium interactions and could play a role in the abnormal adhesion of SS RBCs to vascular endothelium contributing to the vaso-occlusive crises reported for sickle cell disease patients.
Asunto(s)
Anemia de Células Falciformes/fisiopatología , Moléculas de Adhesión Celular/fisiología , Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Eritrocitos/fisiología , Proteínas de Neoplasias/fisiología , Anemia de Células Falciformes/sangre , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/biosíntesis , Epinefrina/farmacología , Eritrocitos/efectos de los fármacos , Humanos , Integrina alfa4beta1/fisiología , Sistema del Grupo Sanguíneo Lutheran , Proteínas de Neoplasias/biosíntesisRESUMEN
Advanced glycation end-products (AGEs) inhibit ischemia-induced angiogenesis but are potential triggers of neoangiogenesis that occurs in peritoneal dialysis (PD) patients. We investigated whether the effect of glucose and AGEs on human peritoneal mesothelial cells (HPMCs) might alter the release of vascular endothelial growth factor (VEGF) and subsequently the formation of capillary tubes by human umbilical vein endothelial cells (HUVECs). HPMCs were exposed to glucose and the glycated protein Nvarepsilon-(carboxymethyl)lysine-human serum albumin (CML-HSA) and VEGF production was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Capillary tube formation by HUVECs in presence of HPMC supernatant or co-cultured with HPMC was investigated. AGE and VEGF levels in PD effluents from 11 patients were measured. CML-HSA stimulated VEGF production by HPMCs, P<0.001. Glucose and AGE inhibited capillary tube formation by HUVECs, P<0.001. HPMC supernatant potentiated capillary tube formation, P<0.001. In co-culture with HPMC capillary tube formation was increased, especially by HPMCs stimulated by CML-HSA, P<0.001. Anti-VEGF antibody limited this effect, P<0.001. Preincubation of HPMCs with anti-receptor for AGEs (RAGE) antibody reduced capillary tube formation, P<0.001. AGE and VEGF levels in PD effluents were increased during long dwell time, P<0.05 and P<0.001, respectively. In a co-culture system, we showed that VEGF production by HPMC favors capillary tube formation through mesothelial RAGE activation and could explain neoangiogenesis in PD patient.
Asunto(s)
Endotelio Vascular/fisiología , Productos Finales de Glicación Avanzada/farmacología , Neovascularización Fisiológica , Peritoneo/efectos de los fármacos , Receptores Inmunológicos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Anticuerpos/farmacología , Capilares/crecimiento & desarrollo , Técnicas de Cocultivo , Endotelio/citología , Endotelio/efectos de los fármacos , Endotelio Vascular/citología , Glucosa/farmacología , Humanos , Lisina/análogos & derivados , Lisina/farmacología , Diálisis Peritoneal , Peritoneo/citología , Receptor para Productos Finales de Glicación Avanzada , Albúmina Sérica/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Glucose or glucose derived products are increased in blood during the postprandial phase and are, to a certain extent, related to meal composition. Glucose and glucose derived products such as advanced glycation end products (AGEs) can be formed in the intracellular compartment but can also be absorbed as AGEs or AGE precursors present in food. Glucose, glucose metabolites and AGEs alter endothelial cell functions, induce adhesion molecule overexpression (ICAM-1, VCAM), cytokine release (IL-6, MCP-1) and tissue factor production. Tumor necrosis factor alpha systemic level is increased during the postprandial phase as are augmented C reactive protein and fibrinogen level. Hyperglycemia induced an increase in plasminogen activator inhibitor, and shortened fibrinogen half life. Hyperglycemia and AGEs provoked an oxidant stress. The formation of reactive oxygen intermediates perturbates NO (Nitric oxide) formation and are deleterious for cell functions. All the modifications observed in the postprandial phase are not too deleterious but their iterative characteristics may lead to vascular dysfunction.
Asunto(s)
Hemostasis , Hiperglucemia/fisiopatología , Inflamación/fisiopatología , Estrés Oxidativo , Periodo Posprandial , Arginina/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Lisina/metabolismoRESUMEN
AIMS: Hyperglycemia is linked to vascular dysfunction in patients with diabetes mellitus, either directly or through advanced glycation end product (AGE) formation. Experimental evidence has indicated the possible involvement of AGEs in the genesis of vascular complications. We investigated whether serum levels of AGEs and of the glycoxidation compound carboxymethyl-lysine (CML) were increased and correlated with vascular complications in type II diabetes mellitus. METHODS: Serum levels of AGEs and CML-human serum protein (CML-HSP) were measured by a specific immunoassay in 51 men and 26 women aged 58 +/- 6.1 years (mean +/- SD) who had been treated for type II diabetes mellitus for 11 +/- 8 years, and in a non-diabetic control group consisting of 39 men and 21 women aged 55.5 +/- 7.5 years. Patients with macroalbuminuria or abnormal creatinine clearance were excluded from the study. RESULTS: The serum levels of AGEs were significantly increased in patients with type II diabetes compared to controls (P<0.001). Blood levels of CML-HSP were significantly increased in diabetic patients compared to normal subjects [35.3 +/- 27.4 and 9.3 +/- 7.2 (mean +/- SD) pmol/mg of protein, respectively; P<0.0001]. In diabetic patients with retinopathy or microalbuminuria (urinary albumin excretion: UAE > 30 mg/24 h), CML-HSP levels were significantly higher (P<0.02), and even more elevated in patients with both complications. CONCLUSION: In patients with type II diabetes, CML-HSP levels that are at variance with the HbA(1c) index for blood glucose may be a biomarker of glycoxidation, and related to the development of microvascular complications.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Angiopatías Diabéticas/diagnóstico , Productos Finales de Glicación Avanzada/sangre , Lisina/análogos & derivados , Lisina/sangre , Microcirculación/fisiología , Biomarcadores/sangre , Glucemia/metabolismo , Índice de Masa Corporal , Angiopatías Diabéticas/sangre , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Leucocyte adhesion is an important phenomenon in antimicrobial defence, inflammation and immunological mechanisms and has been shown to be dependent upon specialized adhesion molecules. To prevent side-effects related to blood transfusion (e.g. anti-human leucocyte antigen immunization and transmission of infectious agents) leucocyte reduction of blood products is now systematically performed in various countries. The most common system used for leucoreduction is blood filtration. For further understanding of the mechanisms responsible for the interaction between leucocytes and the fibres present in filters we used a flow chamber to study the adhesion of leucocytes and leukaemic cell lines to different types of fibre. Adhesion was quantified using video-microscopy and computer image analysis. Our results demonstrate that adhesion to filter fibres was dependent on the expression of beta2-integrins CD11--CD18 and was inhibited by anti-CD18. The amount of fibres present, their spatial arrangement and the physicochemical characteristics of the fibres were important factors in leucocyte adhesion. Leucocyte adhesion was the highest to polyethylene terephthalate (PET) and polyimide fibres. Lymphocytes or lymphocytic cell lines were poorly adherent to PET fibres. The retaining capacity of leucocyte filters can be improved by taking into account the different parameters for the design of new filters
Asunto(s)
Alquenos , Leucaféresis/instrumentación , Leucemia/inmunología , Leucocitos/fisiología , Filtros Microporos , Anticuerpos Monoclonales/farmacología , Antígenos CD18/inmunología , Antígenos CD18/fisiología , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/fisiología , Celulosa , Citometría de Flujo , Granulocitos/química , Granulocitos/fisiología , Células HL-60 , Humanos , Procesamiento de Imagen Asistido por Computador , Integrina alfaXbeta2/fisiología , Selectina L/fisiología , Leucaféresis/métodos , Leucocitos/química , Antígeno-1 Asociado a Función de Linfocito/fisiología , Linfocitos/química , Linfocitos/fisiología , Antígeno de Macrófago-1/fisiología , Microscopía por Video , Monocitos/química , Monocitos/fisiología , Poliésteres , Tereftalatos Polietilenos , Polipropilenos , Alcohol Polivinílico , Células Tumorales Cultivadas/inmunologíaRESUMEN
Engagement of the receptor for advanced glycation end products (RAGE) by products of nonenzymatic glycation/oxidation triggers the generation of reactive oxygen species (ROS), thereby altering gene expression. Because dissection of the precise events by which ROS are generated via RAGE is relevant to the pathogenesis of complications in AGE-related disorders, such as diabetes and renal failure, we tested the hypothesis that activation of NADPH oxidase contributed, at least in part, to enhancing oxidant stress via RAGE. Here we show that incubation of human endothelial cells with AGEs on the surface of diabetic red blood cells, or specific AGEs, (carboxymethyl)lysine (CML)-modified adducts, prompted intracellular generation of hydrogen peroxide, cell surface expression of vascular cell adhesion molecule-1, and generation of tissue factor in a manner suppressed by treatment with diphenyliodonium, but not by inhibitors of nitric oxide. Consistent with an important role for NADPH oxidase, although macrophages derived from wild-type mice expressed enhanced levels of tissue factor upon stimulation with AGE, macrophages derived from mice deficient in a central subunit of NADPH oxidase, gp91phox, failed to display enhanced tissue factor in the presence of AGE. These findings underscore a central role of NADPH oxidase in AGE-RAGE-mediated generation of ROS and provide a mechanism for altered gene expression in AGE-related disorders.
Asunto(s)
Regulación de la Expresión Génica , Productos Finales de Glicación Avanzada/farmacología , NADPH Oxidasas/metabolismo , Estrés Oxidativo , Receptores Inmunológicos/fisiología , Animales , Células Cultivadas , Diabetes Mellitus Tipo 1/sangre , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Eritrocitos/metabolismo , Productos Finales de Glicación Avanzada/sangre , Humanos , Peróxido de Hidrógeno/metabolismo , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/deficiencia , Ratones , NADPH Oxidasa 2 , Receptor para Productos Finales de Glicación Avanzada , Tromboplastina/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The diabetic vasculopathy is one of the major complications responsible for the high incidence of arteriopathy, coronary ischemia and renal failure. Several hypothesis have been formulated to explain the vascular abnormalities. We recently showed that advanced glycation end products (AGE) have a pivotal role in the genesis of vascular dysfunction. AGE bind to a receptor (RAGE) present on endothelial cells. AGE binding to RAGE produced an oxidant stress and diminished vascular barrier function, increased vascular permeability, enhanced the expression of vascular cell adhesion molecule 1 (VCAM-1). VCAM-1 expression on endothelial cell and increased expression of CD11b CD18 on monocytes may facilitate monocyte emigration and can represent one of the initial steps of vascular alteration. In diabetic animals or in ApoE null diabetic mice which developed atherosclerosis, the infusion of recombinant RAGE prepared in insect cells was studied. Recombinant RAGE administration corrected vascular hyperpermeability and prevented the development of atherosclerosis in the animals.
Asunto(s)
Células Sanguíneas/patología , Angiopatías Diabéticas/sangre , Endotelio Vascular/patología , Animales , Células Sanguíneas/fisiología , Comunicación Celular/fisiología , Endotelio Vascular/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Productos Finales de Glicación Avanzada/fisiología , Humanos , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/fisiología , Enfermedades Vasculares/etiología , Enfermedades Vasculares/patologíaRESUMEN
BACKGROUND: Blood filtration is a technique widely used to reduce the levels of WBCs in blood components. Several studies have been conducted to define the factors that are involved in WBC reduction, but the various mechanisms are not clearly delineated. This study explored the role of WBC adhesion molecules in WBC reduction during filtration. STUDY DESIGN AND METHODS: A minifilter has been developed that has properties similar to those of the standard filter (Sepacell, Asahi Medical) but that allows a smaller volume of blood to be used (15 mL). WBC reduction was achieved to a similar extent in the standard filter and the minifilter (4.15 log and 4.18 log, respectively). Samples of human promyelocytic cell line (HL60) were filtered before and after differentiation induced by vitamin D3 (D3-HL60). Flow cytometry was used to characterize the D3-HL60 filtrates and to count the WBCs after filtration. RESULTS: HL60 was retained in the filter to the same extent as all other WBCs. A higher level of integrin receptors (CD11b/CD18; CD11c/CD18) was expressed by D3-HL60 than by HL60. When the blood was incubated with anti-CD11b, anti-CD11c, or anti-CD18, fewer D3-HL60 cells were trapped by the filter, while only anti-CD11b alters HL60 retention in the filter. CONCLUSION: The receptors CD11b/CD18 and CD11c/CD18 appear to bind to the filter fibers and to be one of the mechanisms responsible for WBC retention.
Asunto(s)
Células HL-60 , Plaquetas/citología , Recuento de Células , Diferenciación Celular , Filtración/instrumentación , Células HL-60/citología , Hemofiltración/métodos , Humanos , LeucaféresisRESUMEN
OBJECTIVE: Cell adhesion phenomenon has been extensively studied in the last decade and was shown to be mediated by specialized molecules and driven by physical forces. Cohesion of the vessel wall cells is also dependent on adhesion molecules but less is known about the physical forces involved. To investigate endothelial cell/endothelial cell interaction from a mechanical point of view, we have used an ultrasonic interferometry device, named EchoCell, which has been previously designed to study red blood cell-red bood cell (RBC-RBC) interaction. METHODS: Bovine aortic endothelial (BAE) cells were cultured, detached, then suspended in buffer and their mechanical and geometrical properties studied with the EchoCell system. The ultrasonic apparatus measures both the accumulation rate of cells in suspension on a solid plate and the acoustical impedances of the suspension and the sediment. RESULTS: In suspension, BAE exhibited, in our experimental conditions (3x10(6) cells per ml), a spherical size evaluated by calculation at a mean radius of 7+/-2 microm. Moreover, no BAE aggregation occurred at the concentrations used. The acoustical impedance of the BAE suspensions calculated from all the samples studied, in the cell concentration range from 1.5x10(6) to 6x10(6) cells per ml, was 1.52x10(6) Rayl (kg m(-2) s(-1)). Furthermore, the acoustical impedance of the cell sediment was found to be independent on the initial cell suspension concentration and equal to 1.63x10(6) Rayl (kg m(-2) s(-1)). Estimation of the volume fraction of BAE inside the sediment allows to evaluate the ultrasonic velocity and the elastic bulk modulus of cells. CONCLUSION: The ultrasonic interferometry method appears particularly interesting to study geometrical and mechanical (acoustical impedance, sound velocity, elastic bulk modulus) properties of BAE cells.
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Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Interferometría/métodos , Animales , Aorta/citología , Bovinos , Movimiento Celular , Tamaño de la Célula , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/diagnóstico por imagen , Reproducibilidad de los Resultados , UltrasonografíaRESUMEN
A human endothelial cell line is a convenient tool for exploring cell physiology and testing drugs and toxics. Several attempts have been made using SV40 to immortalize endothelial cells. We used human umbilical vein endothelial cells (HUVEC) transformed with a construct made of promoter of the vimentin gene and SV40 Tag. The proliferation of immortalized vascular endothelial cells (IVEC), as measured by [methyl-3H]thymidine incorporation, was compared to that of HUVEC in the presence of endothelial cell growth factor and cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma). Inhibition of [methyl-3H]thymidine incorporation by IL-1beta was lower than that observed with HUVEC, while TNF-alpha reduced the proliferation of IVEC and HUVEC to similar extents. Induction of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin by TNF-alpha, measured by a radiometric technique, was similar in IVEC and HUVEC, while the induction of E-selectin by IL-1beta on IVEC was limited and significantly different from that observed on HUVEC (p<0.001). The number of 125I-IL-1beta binding sites on IVEC is 3-fold less than on HUVEC and the IL-1beta receptor number was reduced. Dexamethasone treatment of IVEC restored their reactivity to IL-1beta and corrected the IL-1beta binding and the receptor number. These results showed that the introduction of SV40 gene not only immortalized the cell but also altered IL-1 receptor expression. This alteration may be improved by addition of corticosteroids to the cell culture, which extends the possibility of using IVEC as a model of endothelial cells.
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Dexametasona/farmacología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Glucocorticoides/farmacología , Interleucina-1/farmacología , Receptores de Interleucina-1/biosíntesis , Línea Celular Transformada , Selectina E/biosíntesis , Endotelio Vascular/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Interferón gamma/farmacología , Receptores de Adhesión de Leucocito/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
Vascular malformations are frequent in newborns, and they persist throughout life, which differentiates them from vascular tumors (eg, hemangiomas). Arteriovenous malformations are high-flow vascular malformations. They are considered nonmalignant but can expand and become a significant clinical risk when extensive. To characterize endothelial cells from arteriovenous malformations (AMEC), we cultured cells obtained from surgical specimens and studied their properties. After selection, the cells that grew out from explants had phenotypic and antigenic features (platelet endothelial cell adhesion molecule, von Willebrand factor) of human endothelial cells. Their spontaneous proliferation rate was higher (1.8 to 6.4 times) than that of human umbilical vein, arterial, or microvascular endothelial cells. The proliferation rate of AMEC was not sensitive to the inhibitory activity of various cytokines (interleukin-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, Interferon-gamma). In basal conditions, intercellular adhesion molecule (ICAM-1) was detected at a higher level of expression (6- to 10-fold) on AMEC, but these cells failed to express E-selectin or the vascular cell adhesion molecule (VCAM-1) after cytokine stimulation. Expression of c-ets-1 proto-oncogene was shown by in situ hybridization. The low response to cytokines, the higher propensity to proliferate, and the ets-1 expression suggest that AMEC have a defective regulation of proliferation that may be due to a reduced apoptotic process.
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Malformaciones Arteriovenosas/patología , División Celular/fisiología , Citocinas/farmacología , Endotelio Vascular/patología , Adolescente , Adulto , Malformaciones Arteriovenosas/cirugía , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Medios de Cultivo Condicionados , Selectina E/genética , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Fibroblastos , Citometría de Flujo , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Masculino , Técnicas de Cultivo de Órganos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Proto-Oncogenes Mas , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Arterias Umbilicales , Venas Umbilicales , Molécula 1 de Adhesión Celular Vascular/genética , Factor de von Willebrand/análisisRESUMEN
The accelerated formation of advanced glycation end products (AGEs) is implicated in diabetic microvascular and macrovascular complications. The binding of AGEs to their cellular surface receptor (RAGE) induces vascular dysfunction and in particular an increase in vascular permeability. We previously demonstrated that rat recombinant RAGE (rR-RAGE) produced in insect cells corrected the hyperpermeability due to RAGE-AGE interaction and that pharmacokinetic properties of rR-RAGE after i.v. administration in rats were compatible with a potential therapeutic use. In the present study, we showed that recombinant human RAGE (rH-RAGE) had a similar efficacy in inhibiting AGE-induced endothelial alteration and in reducing the hyperpermeability observed in streptozotocin-induced diabetic rats. (125)I-rH-RAGE elimination half-life after i.v. administration was similar in diabetic and normal rats (53.7 +/- 7.6 and 45.3 +/- 4.0 h, respectively). The presence of AGEs is responsible for a higher distribution volume in diabetic rats compared with normal rats (15.3 +/- 2.7 and 7.7 +/- 0. 7 l/kg, respectively). Immunoreactive (125)I-rH-RAGE decreased more rapidly than did immunoreactive (125)I-rR-RAGE. The differences between (125)I-rH-RAGE and (125)I-rR-RAGE pharmacokinetics in rat may be related to differences in potential O-glycosylation and protease cleavage sites between the two RAGE molecules.
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Productos Finales de Glicación Avanzada/metabolismo , Receptores Inmunológicos/metabolismo , Albúminas/farmacocinética , Secuencia de Aminoácidos , Animales , Aorta/metabolismo , Permeabilidad Capilar/fisiología , Bovinos , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Ratas , Ratas Wistar , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Homología de Secuencia de Aminoácido , Distribución Tisular , Ácido Tricloroacético/farmacologíaRESUMEN
The high incidence of vascular complications in patients with diabetes mellitus prompted us to study the pathophysiology of diabetic angiopathy. Hyperglycaemia is a common feature resulting in several metabolic and endocrine alterations and the formation of advanced glycation end-products (AGE). AGE bind to different molecules and to a receptor (RAGE). RAGE interaction with AGE enhances receptor expression and initiates a feedback loop whereby RAGE occupancy triggers increased RAGE expression. In a model of accelerated atherosclerosis associated with diabetes in genetically-manipulated mice, the blockade of cell surface RAGE by infusion in a soluble truncated form completely suppressed enhanced formation of vascular lesions. Improvement of atherosclerosis in these diabetic-atherosclerotic animals through the use of soluble RAGE occurred in the absence of changes in plasma lipids or glycaemia, which emphasises the contribution of a lipid- and glycemia-independent mechanism to atherogenesis.