Background: Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), of nuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSH concentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs). Methods and Results: 52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokers we demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and in peripheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showed impairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECs exposed to smokers’ serum but not to non-smokers’ serum we found that oxidative stress increased, whereas nitric oxide and GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamatecysteine ligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesis that the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposed HUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found in smokers’ serum) the expression of Nrf2/ARE pathway was reduced. The knockdown of Nrf2 was associated to a significant reduction of HO-1 and GCLC expression induced by oxPAPC in ECs. Conclusions: In young smokers with ED a novel further consequence of increased oxidative stress is a repression of Nrf2/ ARE pathway leading to GSH depletion.
CIGARETTE SMOKING BLOCKS THE PROTECTIVE EXPRESSION OF Nrf2/ARE PATHWAY IN BLOOD CELLS OF YOUNG HEAVY SMOKERS FAVOURING INFLAMMATION: RELATION WITH ENDOTHELIAL FUNCTION AND CAROTID INTIMA MEDIA THICKNESS
MOZZINI, Chiara
2009-01-01
Abstract
Background: Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), of nuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSH concentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs). Methods and Results: 52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokers we demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and in peripheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showed impairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECs exposed to smokers’ serum but not to non-smokers’ serum we found that oxidative stress increased, whereas nitric oxide and GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamatecysteine ligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesis that the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposed HUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found in smokers’ serum) the expression of Nrf2/ARE pathway was reduced. The knockdown of Nrf2 was associated to a significant reduction of HO-1 and GCLC expression induced by oxPAPC in ECs. Conclusions: In young smokers with ED a novel further consequence of increased oxidative stress is a repression of Nrf2/ ARE pathway leading to GSH depletion.
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