NO Deficiency and HIF-1a6 Stabilization in HUVECs Figure 2. The enhancement in HUVEC migration induced by L-NAME is reverted by the NO donor DETA-NO and is independent of the cGMP pathway. (A) HUVECs were treated for 48 h with 5 mM L-NAME in the absence or in the presence of 500 nM DETA/NO for the last 24 h, as indicated. Chemotaxis experiments were then performed using 25 ng/ml VEGF as attractants. Results are expressed as the number of migrating cells. #p,0.001 vs basal migration in control cells (CTRL); 1p,0.01 vs VEGF-induced migration in control cells; ***p,0.001 vs basal migration in L- NAME treated cells; uuup,0.001 vs VEGF-induced migration in L-NAME treated cells; no significant differences between control and DETA/NO treated cells (One-way ANOVA with Bonferroni’s test, n = 15). (B) HUVECs were treated for 48 h with 5 mM L-NAME or 1 mM ODQ, and chemotaxis experiments were performed as described in (A). Results are expressed as the number of migrating cells in the different experimental conditions. #p,0.001 vs basal migration in control cells (CTRL); 1p,0.001 vs VEGF-induced migration in control cells; no significant differences between control and ODQ treated cells (One-way ANOVA with Bonferroni’s test, n = 3). (C) cGMP accumulation in HUVECs treated for 48 h with L-NAME or ODQ was evaluated by EIA and expressed as pmol of cGMP normalized to the cell protein content (pmol/mg protein). ***p,0.001; One-way ANOVA with Bonferroni’s test; n = 3. doi:10.1371/journal.pone.0029680.g002 hypoxia-inducible factor-1a (HIF-1a), which plays a major role in the transcriptional activation of genes encoding angiogenic factors [18,19]. Similarly, induction of VEGF expression during hypoxia has been described also in endothelial cells [20]. We therefore analysed the effect of long term L-NAME treatment on HIF-1a levels in HUVECs. Most interestingly, we observed that, after 48 h of treatment, L-NAME induced nuclear accumulation of HIF-1a in HUVECs (5.561.6 fold over basal) (Figures 4A and B). RT- qPCR analysis revealed no significant change in HIF-1a mRNA levels after L-NAME treatment (1.2160.1 fold in comparison to untreated cells) (Figure 4C), suggesting that HIF-1a accumula- tion in L-NAME-treated cells was mainly due to its stabilization, as occurs under hypoxic conditions. Taken together, these data suggest that prolonged L-NAME treatment induces in endothelial cells a pseudohypoxic state, with the consequent stabilization of HIF-1a under normoxic conditions. In addition to HIF-1a, other factors could contribute to the up- regulation of VEGF expression [21]. To directly correlate the increase in VEGF expression induced in HUVECs by L-NAME treatment to the transcriptional activity of HIF-1, we performed experiments in which L-NAME treated cells and control cells were transfected with a plasmid expressing a dominant negative form of the HIF-1b subunit (DARNT), which maintains the capacity of forming an heterodimer but cannot bind DNA [22,23]. As shown in Figure 4D, in HUVECs, the increase in VEGF protein induced by L-NAME treatment was totally blunted, confirming the central role of HIF-1a as regulator of VEGF expression in NO-deprived endothelial cells. Moreover, as shown in Figure 4E, transfection with DARNT inhibited also the increased migratory capacity induced in HUVECs by L-NAME treatment. The NO donor DETA-NO reverts the effects of L-NAME treatment on HIF-1a stabilization, and on VEGF and eNOS expression To investigate whether the observed effects of L-NAME treatment on HUVEC gene expression were due to the chronic depletion of NO, we reconstituted NO levels by utilizing the long lasting NO donor DETA-NO. The addition of 500 nM DETA- NO completely reverted the stabilization of nuclear HIF-1a, the reduced expression of total eNOS protein (Figure 5A) and the increase in VEGF mRNA levels (Figure 5B), which are all characteristic features detected in the long-term L-NAME treated cells. Silencing of eNOS mimics the effects of long-term L- NAME treatment of HUVECs To further investigate if the observed effects of L-NAME treatment were due to the specific inhibitory effect on eNOS activity, expression of the enzyme was silenced in HUVECs by PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29680