NO Deficiency and HIF-1a6 Stabilization in HUVECs Figure 1. Effect of chronic NO deprivation on HUVEC vitality and mitochondrial mass and function. (A) Lysates of HUVECs treated for 48 h with 5 mM L-NAME or 30 mM glucose (high glucose, h-Glc) were separated by 12% SDS-PAGE and immunoblotted with an anti-caspase 3 antibody which recognized full length caspase-3 (35 kDa) and its large fragment resulting from cleavage (17 kDa). b-actin was used as a loading control. Shown is a representative blot of 2 comparable experiments. (B) Total cell lysates prepared as described in (A) were separated by SDS-PAGE and immunoblotted with anti Bcl–2 or anti Bax antibodies. b-actin was used as a loading control. Shown is a representative blot of 2 comparable experiments. (C) Mitochondrial DNA (mtDNA) was quantified by RT-qPCR from control cells (CTRL) or from cells treated with L-NAME for 48 h, and normalized to the level of the housekeeping gene 18S. **p,0.01; t test; n = 3. (D) Mitochondrial activity of control and L-NAME treated cells was evaluated by means of MTS. In parallel samples, the total cell number was measured by crystal violet staining. ***p,0.001 vs control cells (CTRL, set at 100%); t test; n = 7. (E) After L-NAME treatment, oxygen consumption was reduced by 2566% in comparison to control cells. The values were normalized to the cell protein content. *p,0.05; t test, n = 3. (F) Total cellular ATP levels were reduced by 2567% after 48 h of 5 = mM L-NAME treatment. **p,0.01 vs control cells (CTRL, set at 100%); t test; n = 8. doi:10.1371/journal.pone.0029680.g001 comparison to untreated cells. Similarly, KDR protein level in cells chronically treated with L-NAME is 1.8-fold increased as compared to untreated cells (Figure 3E), in good agreement with the RT-qPCR data. A major VEGF signaling pathway involved in endothelial cell migration includes the activation of phosphatidylinositol-3-kinase (PI-3-K) and the subsequent phosphorylation of the protein kinase AKT, which in turn activates eNOS by phosphorylating it on Ser1177. To determine if L-NAME treatment modified the ability of VEGF to activate this pathway, the phosphorylation state of eNOS and AKT after a 5-min VEGF stimulation in control and chronically L-NAME treated cells was measured. As shown in Figure 3F, in control cells VEGF (25 ng/ml) increased eNOS and AKT phosphorylation by about 3 times, as expected (lane 2). In L-NAME treated cells, the basal levels of eNOS and AKT phosphorylation were already increased (see lane 3 vs lane 1), and VEGF was not able to induce any further phosphorylation (lane 4). A densitometric analysis performed on 4 independent experiments revealed that in L-NAME treated cells the basal level of phosphorylated eNOS was 3.4360.94 times greater than in control cells. The increase was less pronounced when the basal level of phosphorylated AKT was compared in treated and control cells (1.5760.24 times). The results presented in Figs. 3 C-F are consistent with an activated VEGF/KDR system in L-NAME-treated HUVECs, and could explain the enhancement of both basal and VEGF- stimulated chemotactic response in these cells. Inhibition of basal NO production induces nuclear HIF-1a accumulation in HUVECs Increased VEGF production and cell motility are typical events occurring in hypoxic cancer cells, due to the accumulation of PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29680