RESUMEN
Circulating endothelial progenitor cells (EPCs) promote vascular repair and maintain integrity of the endothelial monolayer. Reduced EPCs number has been associated with endothelial dysfunction in various cardiovascular diseases. Cardiovascular disease risk is higher in renal transplant patients (RT) than the general population. We studied EPCs number and proliferation in RT, and examined the association with other cardiovascular risk factors such as reduced glomerular filtration rate (GFR) and LDL cholesterol. EPCs concentration was determined in 94 RT and 39 control subjects (C) by flow cytometry. EPCs proliferation was also studied after 7 days in culture. EPCs concentration was significantly reduced in RT versus C (median 33.5 [5-177] vs. 53 [9-257] EPCs/10(5) PMN cells, p=0.006). EPCs proliferation was also reduced in RT versus C (mean+/-SD; 372.7+/-229.3 vs. 539.8+/-291.3 EPCs x field, p=0.003). In multiple regression analysis, GFR, HDL, LDL and body weight were independent predictors of EPCs concentration in RT (r2=0.25, p<0.001). EPCs number is reduced in RT, particularly in patients with reduced GFR. Moreover, EPCs from RT studied in vitro, showed reduced proliferation, which is a sign of functional impairment. These alterations may be involved in increased cardiovascular risk of RT.
Asunto(s)
Células Endoteliales/citología , Trasplante de Riñón , Células Madre/citología , Antígeno AC133 , Adulto , Antígenos CD/biosíntesis , Antígenos CD34/biosíntesis , Peso Corporal , Enfermedades Cardiovasculares/patología , Sistema Cardiovascular/patología , Proliferación Celular , Femenino , Citometría de Flujo , Tasa de Filtración Glomerular , Glicoproteínas/biosíntesis , Humanos , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Fallo Renal Crónico , Antígenos Comunes de Leucocito/biosíntesis , Modelos Lineales , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Péptidos , Análisis de Regresión , Factores Sexuales , Factores de TiempoRESUMEN
OBJECTIVE: The low number of postnatal endothelial progenitor cells (EPC) in the circulation limits their therapeutic application in cardiovascular medicine. Processed lipoaspirate (PLA) cells differentiate into osteoid, adipose, muscle, and cartilaginous cells. This study examines the potential of PLA cells as a source of EPCs. METHODS: PLA cells obtained from human lipoaspirates were cultured for 1 week in serum-depleted medium to form three-dimensional cell clusters (3DCC). The phenotype of 3DCC-derived cells was assessed by immunofluorescence staining and FACS analysis. RESULTS: Flow cytometry showed that 45+/-5% of cells derived from the 3DCC expressed Flk-1, a marker of early EPC, whilst only 4+/-0.5% of freshly isolated PLA were Flk-1+. The proportion of Flk-1+ cells increased to 98+/-2% during culture in hematopoietic stem cell medium. When cultured in an endothelial cell (EC)-specific medium, Flk-1+ cells also expressed Ve-cadherin, von Willebrand's factor (vW), and a lectin receptor, and took up low-density lipoprotein. Incorporation into an endothelial cell tubular network confirmed their functional activity. CONCLUSION: This report describes the first isolation and culture of Flk-1+ cells from human adipose tissue. The feasibility of the extraction and culture of these cells in increased numbers suggests that such autologous cells will be useful for applications ranging from basic research to cell-based therapies.
Asunto(s)
Adipocitos/metabolismo , Células Endoteliales/citología , Células Madre/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Separación Celular/métodos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , LipectomíaRESUMEN
Transforming growth factor-beta (TGFbeta1) is a proangiogenic factor both, in vitro and in vivo, that is mainly involved in the later phases of angiogenesis. In an attempt to identify genes that participate in this effect, we found that TGFbeta1 down-regulates expression of adenylate cyclase VI. In addition, cAMP analogs (8-Bromo-cAMP) and forskolin (an adenylate cyclase activator) also reduced TGFbeta1-induced in vitro angiogenesis in mouse endothelial cell lines and in primary cultures of human umbilical vein endothelial cells on collagen gels. Induction of Ets-1 and plasminogen activator inhibitor-1 (PAI-1) by TGFbeta1 was blocked by these cAMP agonists and activators, in the absence of effects on endothelial cell viability. Moreover, the signal transduction pathways stimulated by TGFbeta1 were unaffected. Thus, Smad2 was normally phosphorylated and translocated to the nucleus in the presence of forskolin. In contrast, transfection studies using the PAI-1-promoter indicated that these cAMP analogues inhibit transcriptional stimulation by TGFbeta1. Electrophoretic mobility shift assay showed that Smad2/3 were bound normally to a TGFbeta1-response region in the presence of the cAMP analogs. In all, these data suggest that the cAMP pathway inhibits the transcriptional activity of Smads, that could be responsible for the block of the TGFbeta1-induced in vitro angiogenesis caused by this second messenger.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/farmacología , AMP Cíclico/farmacología , Endotelio Vascular/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Capilares/efectos de los fármacos , Capilares/fisiología , Línea Celular , Endotelio Vascular/efectos de los fármacos , Humanos , Ratones , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1 , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/fisiologíaRESUMEN
The blood-brain barrier (BBB) ensures the homeostasis of the brain microenvironment, mostly through complex tight junctions between brain endothelial cells that prevent the passage of hydrophilic molecules from blood to brain and vice versa. A recent study has shown in vivo that systemic administration of erythropoietin (Epo) protects against brain injury. Using an in vitro model of the bovine BBB, we observed that the expression of the Epo receptor is modulated by its ligand and hypoxic stimuli such as vascular endothelial growth factor (VEGF) treatment. In addition, Epo protects against the VEGF-induced permeability of the BBB, decreases the levels of endothelial nitric oxide synthase and restores junction proteins. The kinetic transport experiments revealed the capacity of Epo to cross the in vitro BBB in a saturable and specific way. Our results suggest a new mechanism for Epo-induced neuroprotection, in which circulating Epo controls and maintains the BBB through an Epo receptor signalling pathway and the re-establishment of cell junctions.