Figure 5. The NO donor DETA-NO reverts the effects of L- NAME treatment on HIF-1a stabilization, and on VEGF and eNOS expression. (A) HIF-1a protein levels were detected by western blotting of nuclear extracts from HUVECs treated with L-NAME and/or DETA/NO as described in Fig. 2A. An aliquot of total cell lysates was immunoblotted with anti eNOS antibodies, and with anti b-actin antibodies as loading control. A representative blot of 3 comparable experiments is shown. (B) VEGF RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. *p,0.05 vs control cells (CTRL); **p,0.01 vs L-NAME treated cells; no significant differences between control and DETA/NO treated cells (One-way ANOVA with Bonferroni’s test; n = 3). doi:10.1371/journal.pone.0029680.g005 supporting the hypothesis of an impaired PHD activity. The question is then shifted to the mechanism through which NO may affect PHD activity in HUVECs. It is well known that NO competes with O2 for the binding to the heme moiety of cytochrome C oxidase [26–28]. Therefore, a possibility that we considered was that NO deficiency, by activating mitochondrial respiration, could cause an intracellular O2 redistribution from the cytoplasm to the mitochondria [29], thus inducing oxygen depletion and PHD inactivation in the cytosol. However, it is unlikely that cytoplasmic hypoxia could have occurred in our experimental conditions where cells are exposed to an atmospheric O2 concentration (21% O2) [30]. In addition, in NO deficient cells we observed a lower oxygen consumption, which can be explained by the reduced mitochon- drial mass, thus making the hypothesis of an O2 redistribution from the cytoplasm to the mitochondria difficult to support. We conclude therefore that NO deficiency affects PHD activity under normoxic conditions. The relationship between NO and HIF-1a is complex and matter of intense debate [31]. Recent studies have suggested a dual NO Deficiency and HIF-1a6 Stabilization in HUVECs role for NO in regulating HIF-1a function. By using NO donors or the controlled expression of an inducible NOS, it has been found that high concentrations of NO induced HIF-1a nuclear accumulation, even under normoxic conditions, whereas under hypoxic conditions, when HIF-1a levels are already high, low physiological concentrations of NO appear to have the opposite effect [32–37]. The effects of the gas under the latter conditions are similar to those observed here. Further underscoring the similarity, the involvement of soluble guanylate cyclase and cGMP in the low NO-induced reduction of HIF-1a was, like in our migration experiments, excluded [32–34,38]. However, differently from the previously reported results, in our experiments, the ECs were not subjected to hypoxic conditions. It is possible that in ECs, the O2 threshold for HIF-1a stabilization is higher than in other cells, and that under normoxic conditions, basal NO production counteracts the stabilization that would otherwise occur. It should be also considered that PHD activity is affected not only by O2, but also by the availability of iron, ascorbate and the Krebs cycle intermediate 2-oxoglutarate [39]. The deficiency of NO may influence the intracellular availability of one or more of these factors, thus decreasing the enzymatic activity and causing HIF-1a accumulation. We cannot however exclude that other stabilizing mechanisms, not directly linked to PHD, are involved [40]. Furthermore, the possibility that reactive oxygen species may play a role is now under investigation in our laboratory. Certainly, more work is needed to clarify these important points. In our experiments very low concentrations of DETA-NO abrogated the HIF-1a accumulation, the VEGF increased expression and the increased motility found in L-NAME treated cells. On the basis of this result one can hypothesize that low levels of the gas constantly produced by ECs under basal conditions contribute to keep HIF-1a levels under control. In other words, under physiological conditions, the ECs, by producing constitutive NO, could control all of the events set in motion by HIF-1a, including cell motility and VEGF production. In case of NO deficiency, HIF-1a would be released from this brake, would escape degradation and consequently accumulate, as shown in our experiments. In conclusion, our results show that lack of NO in human endothelial cells induces pseudohypoxia and mitochondrial dysfunction with consequent decreased energy production. These events may very well occur in all of those pathological conditions where eNOS expression and/or activity are impaired, as in hypertension, type 2 diabetes, hypercholesterolemia, thus contrib- uting to the endothelial dysfunction typical of such disorders. Our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study the effect of NO deficiency on vascular endothelial cells, and to find new therapeutic target (HIF-1a protein for example) possibly useful for treating endothelial dysfunction. Materials and Methods Cell cultures Human umbilical vein endothelial cells (HUVECs) were isolated from freshly derived umbilical cords by digestion with collagenase as described by Jaffe et al. [41]. Umbilical cords were donated anonymously after informed consent according to national ethical legislation. Cells were routinely grown in 199 medium, supplemented with 20% heat-inactivated fetal bovine serum (FBS), 100 mg/ml endothelial cell growth supplement (ECGS) and 50 mg/ml heparin, and used at passages 2–7. Where indicated, HUVECs were treated with 5 mM NG-Nitro-L-arginine PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29680