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

Abstract

A common cardiovascular complication of diabetes is an increase in the vascular escape rate of albumin, generally interpreted as an increase in microvascular permeability [1±3]. One of the more recent theories to explain this endothelial dysfunction focuses on the accumulation in blood and tissues of the advanced glycation end products (AGEs) formed from protein modification by glucose [4]. The AGE hypothesis holds that chronic diabetes-accelerated chemical modification of proteins, lipids and other molecules by reducing sugars alters their structure and function, inducing cellular Diabetologia (2001) Abstract 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 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 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 mmol/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 (~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-tocell and cell-to-matrix adhesion molecules. Conclusion/interpretation. Trace amounts of redoxactive 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. [Diabetologia (2001

Glycolaldehyde‐modified β‐lactoglobulin AGEs are unable to stimulate inflammatory signaling pathways in RAGE‐expressing human cell lines

“…Thus, it can be concluded that AGEs can only stimulate inflammatory signaling under specific conditions such as the presence of endotoxin or perhaps transition metals [32] but not by themselves. Our data add to this with the demonstration that the commonly used endotoxin removal resins are insufficient to remove the cytokine inducing activity present in most AGE preparations and even some unglycated control proteins.…”

Interaction between GSTM1/GSTT1 Polymorphism and Blood Mercury on Birth Weight

“…Hg has been reported to cause oxidative stress, which may lead to lipid peroxidation and the generation of reactive oxygen species (Huang et al 2008;Pinheiro et al 2008). Hui et al (2001) suggested that heavy metals, such as Hg and cadmium, might induce oxidative stress caused by changes in the GSH and/or ATP metabolism. Also, pregnancy is a condition that favors oxidative stress, mainly due to the mitochondriarich placenta, which may also be enhanced by the presence of metal toxins (Casanueva and Viteriy 2003;Chen and Lin 1998).…”

Binding of receptor for advanced glycation end products (RAGE) ligands is not sufficient to induce inflammatory signals: lack of activity of endotoxin-free albumin-derived advanced glycation end products

“…Various metal ions have been shown to drive inflammatory responses via generation of reactive oxygen species, leading to NF-κB activation [36,37]. Hui et al [38] reported that oxidant stress and endothelial permeability previously attributed to AGE proteins could be induced by redox-active metal ions found in the biological buffers commonly used to generate AGE preparations in vitro.…”