Prolonged exposure to LPS increases iron, heme, and p22phox levels and NADPH oxidase activity in human aortic endothelial cells: inhibition by desferrioxamine.

OBJECTIVE

Vascular oxidative stress and inflammation are contributing factors in atherosclerosis. We recently found that the iron chelator, desferrioxamine (DFO), suppresses NADPH oxidase-mediated oxidative stress and expression of cellular adhesion molecules in mice treated with lipopolysaccharide (LPS). The objective of the present study was to investigate whether and how LPS and iron enhance, and DFO inhibits, NADPH oxidase activity in human aortic endothelial cells (HAECs).

METHODS AND RESULTS

Incubation of HAECs for 24 hours with 5 microg/mL LPS led to a 4-fold increase in NADPH oxidase activity, which was strongly suppressed by pretreatment of the cells for 24 hours with 100 micromol/L DFO. Incubating HAECs with LPS also significantly increased cellular iron and heme levels and mRNA and protein levels of p22phox, a heme-containing, catalytic subunit of NADPH oxidase. All of these effects of LPS on HAECs were strongly inhibited by DFO. Exposing HAECs to 100 micromol/L iron (ferric citrate) for 48 hours exerted similar effects as LPS, and these effects were strongly inhibited by coincubation with DFO. Furthermore, neither LPS nor DFO affected mRNA and protein levels of p47phox a nonheme-containing, regulatory subunit of NADPH oxidase, or the mRNA level of NOX4, an isoform of the principal catalytic subunit of NADPH oxidase in endothelial cells. In contrast, heme oxygenase-1 was strongly suppressed by DFO, both in the absence and presence of LPS or iron.

CONCLUSIONS

Our data indicate that prolonged exposure to LPS or iron increases endothelial NADPH oxidase activity by increasing p22phox gene transcription and cellular levels of iron, heme, and p22phox protein. Iron chelation by DFO effectively suppresses endothelial NADPH oxidase activity, which may be helpful as an adjunct in reducing vascular oxidative stress and inflammation in atherosclerosis.