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.
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 diabeticpatients. However, the exact mechanisms concerning the effects of glycated proteins on vascular tissue remain to be determined. Therefore, the effects of glycated humanserum 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 humanserum 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 humanserum 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 humanserum 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 humanserum albumin protein in diabeticpatients 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: Illa Tea; Gwénaëlle Le Gall; Alice Küster; Nadia Guignard; Marie-Cécile Alexandre-Gouabau; Dominique Darmaun; Richard J Robins Journal: PLoS One Date: 2012-01-23 Impact factor: 3.240