Antioxidant Response of Endothelial Cells to High Glucose

Abstract

Abstract: : Purpose: To evaluate the antioxidant (AOX) arm following oxidative stress in the aqueous and membrane portion of vascular endothelial cells under in vitro conditions of hyperglycemia. Methods: Cultured pulmonary porcine endothelial cells (PECs) and human retinal endothelial cells (HRECs) were used. All cells were grown in low glucose (5 mM) and later subjected to high glucose (25 mM) for 7 days. PECs and HRECs at confluence were trypsinized, centrifuged and pelleted. Meta–phosphoric acid was added to prevent auto–oxidation of glutathione (GSH). Sample buffer, pH 8.0, and o–phthalaldehyde (OPT) were added, incubated at room temperature for 15 min, read flurometrically at 420 nm emmision with excitation at 350 nm, the concentration calculated from a standard curve obtained with the GSH–OPT adduct and expressed in mg/dL. Lipid soluble antioxidants were determined by HPLC on a Supelco LC–18 column. Glutathione peroxidase (GPx) activity using cumene hydroperoxide as substrate and GSH as a cofactor and glutathione reductase (GR) activity using GSH as substrate were measured at 340 nm on a Cobas FARA 2 autoanalyzer and expressed as U/mL of supernate. Results: GSH levels in controls were similar between PECs (0.190 ± 0.061) and HRECs (0.187 ± 0.057) and were unaffected by high glucose. PECs exposed to high glucose showed a decrease in GPx from 0.185 ± 0.078 to 0.140 ± 0.014, but no change in GR activity was observed. ß–carotene was the only carotenoid detected in culture and its level was unchanged after exposure to high glucose. However, treatment did increase the level of α–tocopherol by 35%. δ–tocopherol levels remained unchanged. Conclusions: Since high glucose stimulates the formation of lipid peroxidation products, we now correlate low GPx activity with this observation. This may be due to direct damage by free radicals and/or increased consumption in response to elevated lipid peroxides. At the same time, α–tocopherol is somehow mobilized to scavenge radicals within cell membranes and subcellular organelles. Furthermore, our results demonstrate that both water and lipid soluble AOX, as well as AOX enzymes are present in isolated endothelial cells. Improving GPx activity through targeting lipid peroxides with appropriate AOX supplementation should be explored in vivo to protect the vascular endothelium directly from oxidative stress during neovascularization.

Keywords: antioxidants • pathobiology • vascular cells