Infectious and inflammatory stimuli decrease endothelial nitric oxide synthase activity in vitro

Abstract

Background: Perturbation of iron metabolism, especially the increase of serum ferritin levels, is often associated with both inflammation and hypertension. Changes in iron availability can affect an important regulator of vascular tone, the endothelial nitric oxide synthase (eNOS), activated by a heme-dependent dimerization.

Objective: To study the regulation of the anti-hypertensive eNOS in human endothelial cells, in correlation with iron metabolism alterations and stimuli triggering them in vivo, such as inflammation or infection.

Design: Cells were treated with stimuli mimicking infection or inflammation [lipopolysaccharide (LPS) and/or tumor necrosis factor alpha (TNFalpha)]. and iron shortage (succinylacetone and desferrioxamine). The effect on eNOS expression and activation was evaluated, as well as ferritin content.

Methods: eNOS protein expression was evaluated by separating the monomeric from the active dimeric form by low-temperature sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE), and mRNA was analyzed by semi-quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR). As for LPS and TNFalpha, eNOS monomer decreased already after a 72-h treatment and further at 144 h, whereas the down-regulation of the dimer was slower, peaking at 144 h. Succinylacetone and desferrioxamine were effective only at 144 h. The mRNA levels were increasingly reduced after incubation, more markedly by LPS and TNFalpha together, whereas succinylacetone and desferrioxamine had no effect on transcription. We found that endothelial cells are not the source of increased ferritin production.

Conclusions: The results of this study suggest a down-regulating effect of infectious and inflammatory stimuli on eNOS expression, both at the mRNA level and protein expression or stability and dimerization, enhanced by heme and iron shortage, and indicate eNOS as a possible link between infection and hypertension.