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

• Published in: Diabetologia Vol. 44; no. 10; pp. 1310 - 1317
• Main Authors: HUI, Y. Y, MCAMIS, W. C, BAYNES, J. W, SCHAEFFER, R. C, WOLF, M. B
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01-10-2001; Springer Nature B.V
• Subjects: Actins - analysis; Age; alpha Catenin; Animals; Associated diseases and complications; Biological and medical sciences; Blood; Buthionine Sulfoximine - pharmacology; Cattle; Cell Adhesion; Cell Membrane Permeability; Cells; Cells, Cultured; Chelating Agents - pharmacology; Culture Media, Serum-Free; Cytoskeletal Proteins - analysis; Diabetes; Diabetes. Impaired glucose tolerance; Dose-Response Relationship, Drug; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Endothelium, Vascular - chemistry; Endothelium, Vascular - drug effects; Endothelium, Vascular - physiology; Fluorescent Antibody Technique; Fluorescent Dyes; Glucose; Glutathione - metabolism; Glycation End Products, Advanced - pharmacology; Hypotheses; Kinetics; Medical sciences; Oxidative Stress; Paxillin; Permeability; Phosphates - pharmacology; Phosphoproteins - analysis; Physiology; Proteins; Pulmonary arteries; Pulmonary Artery; Serum Albumin, Bovine - metabolism; Veins & arteries; United States > US; South Carolina; Endocrinopathy; Cell culture; Serum free medium; Oxidative stress; Endothelial cell; Diabetes mellitus; Reaction product; Cardiovascular disease; Ox; In vitro; Glycation; Vascular disease; Vertebrata; Mammalia; Animal; Artiodactyla; Ungulata
• ISSN: 0012-186X; 1432-0428
• DOI: 10.1007/s001250100646

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 studied the effects of AGE-bovine serum albumin on endothelial monolayer permeability and intracellular glutathione.
AGE-BSA was prepared by incubating BSA for 30 days at 37 degrees C 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 endothelial cell monolayer preparation. Glutathione was measured by an enzymatic assay.
AGE-BSA concentrations greater than 3 to 4 micromol/l produced maximal increases in permeability (6-8 times basal) within 3 to 4 h of incubation with the cells. This effect persisted for at least 48 h. However, BSA incubated in the absence of glucose produced similar effects. Dialysis of the AGE-BSA showed that low molecular weight components contained the permeability-increasing activity. Phosphate buffer used to prepare the AGE-BSA, at concentrations equivalent to those present in phosphate-buffered saline and in the AGE preparation (approximately 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 increases. These increases in permeability were associated with a decrease in endothelial glutathione, both inhibited by 10 mmol/l N-acetylcysteine, and a loss of cell-to-cell and cell-to-matrix adhesion molecules.
Trace amounts of redox-active metal ions in biological buffers could induce oxidative stress and alterations in cellular functions attributed to AGE-proteins in vitro. It is important to use metal-free phosphate and bicarbonate buffers in studies on cell biology in vitro, especially in serum-free media.