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

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.

Key structural and functional differences between early and advanced glycation products

“…Interestingly, in our experimental conditions gHSA induced ROS production at extra-and intracellular levels in HUVEC at low concentrations and after 4 h of treatment, as previously shown (Rodino-Janeiro et al 2010). However, AGE-HSA was able to increase extracellular ROS production at lower concentrations and shorter times of treatment than gHSA but only showed a trend to increase intracellular ROS at 25 mg/ml and 4 h of treatment.…”

“…HUVEC were isolated from freshly obtained human umbilical cords donated under informed consent from mothers, following the method previously described (Rodino-Janeiro et al 2010). All procedures were approved by the Ethics Committee for Clinical Research at Galicia (Spain), according to the World Medical Association Declaration of Helsinki.…”

“…Extracellular ROS production of HUVEC was measured by the cytochrome C reduction method, as previously described (Rodino-Janeiro et al 2010). Briefly, after treatments, the HUVEC were incubated in HEPES buffer solution (HBS) supplemented with 1.5 mM CaCl 2 (calcium-rich HBS) containing 200 mM cytochrome C. Superoxide production was induced by adding reduced b-nicotinamide adenine dinucleotide (NADH, 100 mM) to the wells.…”

“…Intracellular ROS production of HUVEC was measured by 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (cDCF-DA; Sigma-Aldrich), as previously described (Rodino-Janeiro et al 2010). Briefly, after treatments, HUVEC were incubated at 37 8C in the dark with EBM-2 medium containing 5 mM cDCF-DA for 15 min.…”

Inibição do estresse oxidativo em macrófagos previne a redução no conteúdo do receptor ABCA-1 induzida por albumina modificada por glicação avançada

“…Os resultados obtidos, a partir das incubações com DPI, apontaram para contribuição da NADPH oxidase na produção de ROS, induzida por albumina-GO e albumina-GAD, em concordância com os achados de outros autores em tipos celulares distintos (Schupp et al, 2005). Além disso, foi demonstrado que a albumina-AGE regula a expressão genética da NADPH oxidase (Rodiño-Janeiro et al 2010). Por sua vez, os experimentos com CCCP evidenciaram contribuição da mitocôndria para o estresse oxidativo celular, induzido por albumina-AGE.…”