RESUMEN
BACKGROUND: Endothelial colony-forming cells (ECFCs) are promising candidates for cell therapy of ischemic diseases. Erythropoietin (EPO) is a cytokine that promotes angiogenesis after ischemic injury. EPO receptors (EPORs) classically include two EPOR subunits, but may also associate with the ß-common chain (CD131) in a newly identified receptor involved in EPO cytoprotective effects. OBJECTIVE: The aim was to take advantage of the proangiogenic properties of EPO to enhance ECFC graft efficiency. We postulated that priming ECFCs by adding epoietin α in culture medium prior to experiments might increase their angiogenic properties. We also explored the role of the CD131 subunit in EPO priming of ECFCs. METHODS AND RESULTS: By western blotting on cord blood ECFC lysates, we showed that EPOR and CD131 expression increased significantly after EPO priming. These proteins coimmunoprecipitated and colocalized, suggesting that they are covalently bound in ECFCs. EPO at 5 IU mL(-1) significantly stimulated proliferation, wound healing, migration and tube formation of ECFCs. EPO priming also increased ECFC resistance to H2 O2-induced apoptosis and survival in vivo. Similarly, in vivo studies showed that, as compared with non-primed ECFC injection, 5 IU mL(-1) EPO-primed ECFCs, injected intravenously 24 h after hindlimb ischemia in athymic nude mice, increased the ischemic/non-ischemic ratios of hindlimb blood flow and capillary density. These effects were all prevented by CD131 small interfering RNA transfection, and involved the phosphoinositide 3-kinase-Akt pathway. CONCLUSION: These results highlight the potential role of EPO-primed ECFCs for cell-based therapy in hindlimb ischemia, and underline the critical role of CD131 as an EPO coreceptor.
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Subunidad beta Común de los Receptores de Citocinas/metabolismo , Endotelio Vascular/efectos de los fármacos , Eritropoyetina/farmacología , Neovascularización Fisiológica , Células Madre/efectos de los fármacos , Animales , Células Cultivadas , Subunidad beta Común de los Receptores de Citocinas/genética , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Células Madre/citología , Células Madre/metabolismo , Regulación hacia ArribaRESUMEN
As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 µg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.
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Células Endoteliales/enzimología , Cetocolesteroles/farmacología , Fluidez de la Membrana/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Supervivencia Celular/efectos de los fármacos , Colesterol/farmacología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Humanos , Lecitinas/farmacología , Peroxidación de Lípido/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Intra-uterine growth restriction (IUGR) is a frequent disease, affecting up to 10% of human pregnancies and responsible for increased perinatal morbidity and mortality. Moreover, low birth weight is an important cause of the metabolic syndrome in the adult. Protein depletion during the gestation of rat females has been widely used as a model for human IUGR. By transcriptome analysis of control and protein-deprived rat placentas, we were able to identify 2543 transcripts modified more than 2.5 fold (1347 induced and 1196 repressed). Automatic functional classification enabled us to identify clusters of induced genes affecting chromosome structure, transcription, intracellular transport, protein modifications and apoptosis. In particular, we suggest the existence of a complex balance regulating apoptosis. Among repressed genes, we noted several groups of genes involved in immunity, signalling and degradation of noxious chemicals. These observations suggest that IUGR placentas have a decreased resistance to external aggression. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites. There was an over-representation of Zn finger (ZNF) proteins and Pdx1 (pancreatic and duodenal homeobox protein 1) putative binding sites. Consistently, Pdx1 and a high proportion of ZNF genes were induced at the transcriptional level. A similar analysis of ZNF promoters showed an increased presence of putative binding sites for the Tata box binding protein (Tbp). Consistently again, we showed that the Tbp and TBP-associated factors (Tafs) were up-regulated in IUGR placentas. Also, samples of human IUGR and control placentas showed that human orthologous ZNFs and PDX1 were transcriptionally induced, especially in non-vascular IUGR. Immunohistochemistry revealed increased expression of PDX1 in IUGR human placentas. In conclusion, our approach permitted the proposition of hypotheses on a hierarchy of gene inductions/repressions leading to massive transcriptional alterations in the IUGR placenta, in humans and in rodents.
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Retardo del Crecimiento Fetal/metabolismo , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Placenta/metabolismo , Regiones Promotoras Genéticas , Adulto , Análisis de Varianza , Animales , Estudios de Casos y Controles , Análisis por Conglomerados , Femenino , Retardo del Crecimiento Fetal/genética , Humanos , Inmunohistoquímica , Recién Nacido , Modelos Animales , Embarazo , Análisis de Componente Principal , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Transcripción GenéticaRESUMEN
OBJECTIVE: To analyze the pattern of adhesion molecules in patients with septic shock. Data sources. - References obtained from Pubmed databank. DATA EXTRACTION: Models of inflammation linking endothelial dysfunction, adhesion molecules and septic states were analyzed. DATA SYNTHESIS: The endothelium has been identified as the central effector in the inflammatory response. Adhesion molecules are strongly involved in the inflammatory process by modulating the leukocyte trafficking. The most important adhesion molecules are the selectins (E-, L-, and P-selectins) and members of the immunoglobulin superfamily (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1). Plasma levels of these molecules are increased in septic shock patients, which may be related to a marked activation of the endothelium. However, a dichotomous profile is observed between plasma and tissue expression. The inhibition of adhesive molecule actions could make it possible to control the inflammatory response.