Effect of advanced glycation end products on oxidative stress in endothelial cells in culture: a warning on the use of cells studied in serum-free media

Aims/hypothesis. Alterations in vascular permeability and oxidative stress are characteristics of endothelial dysfunction in diabetic vascular disease . Since AGE-proteins have been hypothesized to mediate these effects, we st udied the effects of AGE-bovine serum albumin on endothelial monolayer perm eability and intracellular glutathione. Methods. AGE-BSA was prepared by incubating BSA for 30 days at 37 degreesC with 0.5 mol/l glucose and 0.2 mol/l phosphate buffer, pH 7.4. Permeability to fluorescently labelled BSA was assessed in a bovine pulmonary artery en dothelial cell monolayer preparation. Glutathione was measured by an enzyma tic assay. Results. AGE-BSA concentrations greater than 3 to 4 mu mol/l produced maxim al increases in permeability (6-8 times basal) within 3 to 4 h of incubatio n with the cells. This effect persisted for at least 48 h. However, BSA inc ubated in the absence of glucose produced similar effects. Dialysis of the AGE-BSA showed that low molecular weight components contained the permeabil ity-increasing activity. Phosphate buffer used to prepare the AGE-BSA, at c oncentrations equivalent to those present in phosphate-buffered saline and in the AGE preparation (similar to 5 mmol/l), produced similar permeability increases at equivalent incubation times. Metal chelators (0.5 mmol/l) or inclusion of fetal bovine serum (10-20%) blocked these permeability increas es. These increases in permeability were associated with a decrease in endo thelial glutathione, both inhibited by 10 mmol/l N-acetylcysteine, and a lo ss of cell-to-cell and cell-to-matrix adhesion molecules. Conclusion/interpretation. Trace amounts of redoxactive metal ions in biolo gical buffers could induce oxidative stress and alterations in cellular fun ctions attributed to AGE-proteins in vitro. It is important to use metal-fr ee phosphate and bicarbonate buffers in studies on cell biology in vitro, e specially in serum-free media.