Lipopolysaccharide-induced oxidative stress is causally related to increased NADPH oxidase activity; however, the underlying mechanisms are incompletely understood.4,26,31,32
The major findings of this study are that prolonged exposure of human aortic endothelial cells to LPS or iron increases cellular levels of iron, heme, and p22phox
, a heme-containing, catalytic subunit of NADPH oxidase; and treatment of the cells with the iron chelator, desferroxamine, inhibits these effects and prevents the LPS or iron-induced increase in NADPH oxidase activity.
We found that LPS increased NADPH oxidase activity in HAEC in a time- and dose-dependent manner, which is consistent with the observation that LPS dose-dependently increased O2.−
production in human blood vessels.33
Interestingly, our time-course studies showed that LPS caused a small, transient increase in NADPH oxidase activity within the first 30 minutes of incubation (see ), likely due to activation of pre-existing NADPH oxidase. However, this increase was not statistically significant and did not appear to be inhibited by DFO. A significant, sustained increase in NADPH oxidase activity was observed between 12 and 24 hours of incubation with LPS, which was abrogated by DFO. These findings are in agreement with published data that increased O2.−
production by NADPH oxidase required prolonged exposure to LPS, both in vitro
and in vivo
Prolonged exposure to LPS also increased cellular iron, heme, and p22phox
protein levels in HAEC. As a possible explanation for these observations, LPS has been shown to upregulate the divalent metal transporter 1 (DMT1), an iron importer, in bronchial epithelial cells.34
Increased iron uptake supplies cellular iron for heme biosynthesis, which in turn may help stabilize the heme protein, p22phox
We also observed that TNFα increased NADPH oxidase activity in HAEC in a DFO-sensitive manner, and TNFα, like LPS, is known to upregulate DMT1 in bronchial epithelial cells.34
However, although DMT1 is abundant in HAEC, its level was not affected by incubation with LPS (data not shown). Therefore, the mechanism by which LPS stimulates iron uptake into HAEC remains to be fully elucidated.
The above long-term effect of LPS on cellular iron and heme levels may explain why p22phox
protein and NADPH oxidase activity were increased in HAEC after 24 hours of incubation. An additional, major role of iron in NADPH oxidase activity is indicated by the observation that DFO abrogated LPS-induced p22phox
gene transcription, which peaked at around 3 hours of incubation with LPS. It is conceivable that cellular labile (“free”) iron, e.g
., by increasing oxidative stress, plays a critical in LPS or TNFα-induced activation of the redox-sensitive transcription factors, NFκB and AP-1, and subsequent p22phox
In contrast, LPS and DFO had no effect on p47phox
and NOX4 gene expression, suggesting a different mechanism of transcriptional regulation independent of iron.
Incubating HAEC with excess iron, in the form of ferric citrate, mimicked the effects of LPS on cellular iron, heme, and p22phox
protein levels. Ferric citrate was used because the majority of labile iron in humans is found as a complex of ferric iron with citrate.3,39
As discussed above, DFO inhibited the iron or LPS-induced changes in cellular iron, heme, p22phox
, and NADPH oxidase. DFO is transported into cells via endocytosis and remains associated with endosomes,40
from which labile iron is transported to mitochondria for heme biosynthesis.41
Thus, DFO chelates free iron and blocks heme synthesis, which may explain why it affected the cellular level of the heme protein, p22phox
, but not the non-heme protein, p47phox
Neither iron nor LPS affected heme oxygenase-1 in HAEC. Induction of HO-1 has been shown to lower NADPH oxidase activity due to decreased heme availability and destabilization and degradation of p22phox
and cytochrome b558
Hence, our findings presented here and elsewhere26
indicate that LPS and iron increase NADPH oxidase activity independently of HO-1, most likely by upregulating p22phox
gene expression and stabilizing the protein by increasing cellular iron uptake and de novo
synthesis of heme.
Interestingly, DFO blocked HO-1 expression irrespective of the addition of LPS or iron. These data suggest that there is a negative feedback loop between iron and HO-1: chelation of iron with DFO reduces the iron supply for synthesis of heme and stabilization of p22phox, and thus NADPH oxidase activity declines. In turn, this mechanism may negatively regulate HO-1 in order to prevent further degradation of heme and, hence, decreased NADPH oxidase activity. The iron content in HAEC growth media (M199 containing 20% FBS) is about 10 μmol/L, whereas up to 100 μmol/L ferric citrate was added in our experiments to induce an effect on p22phox and NADPH oxidase activity. Nevertheless, the low iron content in the media seems enough to strongly induce HO-1 expression in HAEC, because neither added iron nor LPS further increased HO-1.
In summary, our data show that prolonged exposure to LPS or iron increases endothelial NADPH oxidase activity, in parallel with increased p22phox gene transcription and increased cellular levels of iron, heme, and p22phox protein. All of these effects of LPS and iron were strongly inhibited by the iron chelator, desferrioxamine. Therefore, chelation of excess iron may help attenuate vascular oxidative stress and inflammation and inhibit the development of atherosclerotic vascular diseases.