Chronic deficiency of nitric oxide affects hypoxia inducible factor-1α (HIF-1α) stability and migration in human endothelial cells.
BACKGROUND
Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated.


RESULTS
Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N(G)-Nitro-L-arginine methyl ester (L-NAME) treatment or its expression was down-regulated by RNA interference. After long-term L-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O(2) consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells.


CONCLUSIONS
Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.
Department of Pharmacology, School of Medicine, Università degli Studi di Milano, Milano, Italy.
Maria Grazia
MG
Cattaneo
Elisa
E
Cappellini
Roberta
R
Benfante
Maurizio
M
Ragni
Fausta
F
Omodeo-Salè
Enzo
E
Nisoli
Nica
N
Borgese
Lucia M
LM
Vicentini
D017209:Apoptosis; D002465:Cell Movement; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000235:genetics; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020030:Nitric Oxide Donors; D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester