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.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Hui
Y Y
YY
Department of Pharmacology and Physiology, University of South Carolina School of Medicine, Columbia, South Carolina 29208, USA.
McAmis
W C
WC
Baynes
J W
JW
Schaeffer
R C
RC
Wolf
M B
MB
D000199:Actins / Q000032:analysis; D000818:Animals; D001769:Blood; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002417:Cattle; D002448:Cell Adhesion; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D003598:Cytoskeletal Proteins / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D005978:Glutathione / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D007700:Kinetics; D018384:Oxidative Stress; D051419:Paxillin; D010710:Phosphates / Q000494:pharmacology; D010750:Phosphoproteins / Q000032:analysis; D011651:Pulmonary Artery; D012710:Serum Albumin, Bovine / Q000378:metabolism; D051178:alpha Catenin
D000199:Actins; D002614:Chelating Agents; D016895:Culture Media, Serum-Free; D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; D017127:Glycation End Products, Advanced; D051419:Paxillin; D010710:Phosphates; D010750:Phosphoproteins; D051178:alpha Catenin; D012710:Serum Albumin, Bovine; D019328:Buthionine Sulfoximine; D005978:Glutathione