NO Deficiency and HIF-1a6 Stabilization in HUVECs Figure 4. L-NAME treatment induces HIF-1a nuclear accumulation. (A) HIF-1a protein levels were detected by western blotting of nuclear extracts from control HUVECs (lane 1) or from HUVECs treated with L-NAME for 48 h (lane 2). Shown is a representative blot of 4 comparable experiments. HIF-1a migrates as a doublet with apparent molecular weight of 118 and 120 kDa. (B) Densitometric analysis of nuclear HIF-1a protein levels. *p,0.05; t test; n = 4. (C) HIF-1a RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. No significant differences between control and L-NAME treated cells (t test; n = 3). (D) VEGF protein levels were detected by ELISA measurement in conditioned media collected from HUVECs transfected with the empty vector (pcDNA3) or with the expression vector DARNT, and treated with L- NAME for the 48 h following transfection. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). **p,0.01 vs untreated cells transfected with pcDNA3; ***p,0.001 vs L-NAME treated cells transfected with pcDNA3 (One-way ANOVA with Bonferroni’s test; n = 3). (E) HUVECs were transfected with pcDNA3 or DARNT, and treated with L-NAME for the 48 h following transfection when indicated. Chemotaxis experiments were then performed using 25 ng/ml VEGF as attractant. Results are expressed as the number of migrating cells. #p,0.001 vs basal migration in untreated pcDNA3 cells; 1p,0.001 vs VEGF-induced migration in untreated pcDNA3 cells; ***p,0.001 vs basal migration in pcDNA3 cells treated with L-NAME; uuup,0.001 vs VEGF-induced migration in pcDNA3 cells treated with L-NAME; no significant differences between untreated pcDNA3 and DARNT transfected cells and between untreated and L-NAME treated DARNT tranfected cells (One-way ANOVA with Bonferroni’s test, n = 10). doi:10.1371/journal.pone.0029680.g004 i.e. a state of hypoxia in the presence of normal levels of oxygen. The increased expression of VEGF found in our experiments can be considered a consequence of this pseudohypoxic state since the dominant negative form of the HIF1-b subunit DARNT totally blunted the VEGF protein increase induced by L-NAME treatment. Accordingly, DARNT transfection inhibited also the increased motility in L-NAME treated HUVECs, thus indicating that the migratory behavior we observed in the treated cells can be considered the consequence of the transcriptional activity of HIF- 1a. Of note, the increased HUVEC motility is observed despite a reduction in eNOS levels, and is not induced by chronic treatment with the guanylate cyclase inhibitor ODQ, indicating that cGMP and protein kinase G (PKG) are not involved in the observed effects. Our results open the interesting question of the mechanism by which NO deficiency induces HIF1astabilization in HUVECs. HIF-1a degradation depends on prolyl hydroxylases-catalysed proline hydroxylation, which induces binding of the factor to an ubiquitin ligase (the Von-Hippel Lindau protein) and targets it for proteasomal degradation. The activity of prolyl hydroxylases (PHDs) is dependent on the availability of oxygen and 2- oxoglutarate (a Krebs cycle intermediate) as substrates and on Fe2+ and ascorbate as cofactors. In addition to these mechanisms controlling HIF levels, HIF-1a transcriptional activity can also be negatively controlled via hydroxylation of an asparaginyl residue operated by an O2 dependent enzyme called Factor Inhibiting HIF (FIH-1) [25]. Under hypoxic conditions, PHD and FIH-1 activity are inhibited and HIF-1a accumulates in the nucleus to function as a transcription factor and to evoke adaptive responses to changes in tissue oxygenation. Under our experimental conditions, chronic eNOS inhibition induces HIF-1a nuclear accumulation, apparently as a result of a decreased degradation, PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29680