title: Glycated albumin, a precursor of advanced glycation end-products, up-regulates NADPH oxidase and enhances oxidative stress in human endothelial cells: molecular correlate of diabetic vasculopathy.
abstract: BACKGROUND
  Hyperglycaemia induces non-enzymatic glycation reactions in proteins which generate Amadori products and advanced glycation end-products; the latter are thought to participate in the vascular complications of diabetic patients. However, the exact mechanisms concerning the effects of glycated proteins on vascular tissue remain to be determined. Therefore, the effects of glycated human serum albumin on human umbilical vein endothelial cells were studied.
  
  
  METHODS
  Reactive oxygen species production was measured by the cytochrome C reduction method and by 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (c-DCF-DA) fluorescence after treating human umbilical vein endothelial cells with glycated human serum albumin (6-200 µg/mL). The expression of Nox4 and p22phox mRNAs were analysed by reverse transcription-quantitative polymerase chain reactions and the levels of their proteins were measured by immunofluorescence.
  
  
  RESULTS
  Low concentrations of glycated human serum albumin enhanced reactive oxygen species production in human umbilical vein endothelial cells after 4 h of treatment at both extracellular and intracellular sites. This enhanced production was sustained, although to a lesser extent, after 6 and 12 h of treatment. The gene expression study revealed that Nox4 and p22phox mRNA levels were elevated after 4 h of treatment with glycated human serum albumin. This mRNA elevation and enhanced reactive oxygen species production correlated with an increased expression of the Nox4 protein.
  
  
  CONCLUSIONS
  The results revealed that a circulating and abundant modified glycated human serum albumin protein in diabetic patients induced a sustained reactive oxygen species production in human endothelial cells. This effect may have been due to an up-regulation of Nox4, the main subunit of NADPH oxidase in the endothelium.
authors.affiliation: Departamento de Medicina, Universidad de Santiago de Compostela and Servicio de Cardiología, Complejo Hospitalario Universitario de Santiago de Compostela, Coruña, Spain.
authors.forename: Bruno K
authors.initials: BK
authors.lastname: Rodiño-Janeiro
authors.forename: Mercedes
authors.initials: M
authors.lastname: González-Peteiro
authors.forename: Rafael
authors.initials: R
authors.lastname: Ucieda-Somoza
authors.forename: José R
authors.initials: JR
authors.lastname: González-Juanatey
authors.forename: Ezequiel
authors.initials: E
authors.lastname: Alvarez
mesh_terms: D002478:Cells, Cultured; D003925:Diabetic Angiopathies / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006801:Humans; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000032:analysis; D012709:Serum Albumin / Q000378:metabolism*; D015854:Up-Regulation; D000093423:Glycated Serum Albumin
chemical_list: D017127:Glycation End Products, Advanced; D017382:Reactive Oxygen Species; D012709:Serum Albumin; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C086986:CYBA protein, human; D000093423:Glycated Serum Albumin