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The endothelium explicates its physiological functions by producing active molecules, among which nitric oxide (NO) is particularly important. It is well known that endothelial dysfunction (ED), i.e. an impaired function of the endothelium coupled with a reduced release of NO, is a risk factor for atherosclerosis together with a list of conditions such as hypertension, hypercholesterolemia, smoking, diabetes, and the aging process itself. These conditions are also associated with a significant increase in Reactive Oxygen Species (ROS) in the vascular wall that may contribute to the establishment of ED and to the development of its late effect on cardiovascular system. In the present study, the behavioural and molecular consequences deriving from chronic NO deprivation were investigated in human primary endothelial cells (human umbilical vein endothelial cells, HUVECs). To inhibit NO formation, endothelial nitric oxide synthase (eNOS) was chronically inhibited by treatment with L-NG-Nitroarginine methyl ester (L-NAME), a structural analogue of L-arginine that competitively block the active site of the enzyme, or by transfection with a siRNA specific for eNOS. We observed that a 48-h L-NAME treatment induced in HUVECs a higher migratory capability (evaluated by chemotaxis assays in Boyden?s chamber) which was independent of the reduced activity of the cyclic GMP/protein kinase G pathway present in chronically NO deprived HUVECs. In the attempt to explain the mechanism(s) through which NO deficiency enhances migration, we investigated if chronic L-NAME treatment affected the expression and production of Vascular Endothelial Growth Factor (VEGF) and of its receptor KDR. RT-qPCR analyses, accompanied by ELISA assays and western blot analyses, demonstrated that both VEGF and KDR mRNAs and proteins were significantly augmented in L-NAME treated cells, thus suggesting the establishment of an autocrine loop responsible for the increased migration. Increased VEGF production and cell motility are typical events occurring in hypoxic cancer cells, due to the accumulation of hypoxia-inducible factor-1? (HIF-1?), which plays a major role in the transcriptional activation of genes encoding angiogenic factors. Similarly, induction of VEGF expression during hypoxia has been described in endothelial cells (ECs). Interestingly, we observed a significant nuclear accumulation of HIF-1? in HUVECs chronically treated with L-NAME. Moreover, the transcriptional activity of HIF-1? was responsible for the increases in VEGF/KDR expression and migration since the transfection with ?ARNT (a dominant negative form of the HIF-1? subunit that maintains the capacity of forming an heterodimer but cannot bind DNA) is able to totally blunt both the effects in L-NAME treated HUVECs, thus confirming the involvement of an autocrine loop in the pro-migratory effect induced by NO deprivation. The dependence of HIF-1? stabilization from NO deficiency was confirmed by using the NO donor DETA/NO. Very low doses of DETA/NO reverted both the HIF-1? accumulation and the consequent increases in VEGF expression and cell motility induced by L-NAME treatment. Furthermore, to investigate whether the observed effects were due to the specific inhibitory effect of L-NAME on eNOS activity, we knocked-down the enzyme by using RNA interference methodology. In eNOS silenced cells, HIF-1? accumulated in the nucleus and VEGF production was enhanced thus confirming the dependence of the observed effects on eNOS inhibition. All these results suggest that basal release of NO may act as a negative controller of HIF-1? levels and cell motility in HUVECs with important consequences on ECs physiology. In the attempt to unravel the pathway(s) linking NO deficiency to HIF-1? accumulation and activity, we focus our attention on ROS since their formation has been involved in HIF-1? stabilization in normoxia. We found that acute treatment with L-NAME induced in HUVECs an early and transient burst in ROS formation that was fully prevented by the presence of the antioxidant N-acetylcysteine (NAC). HIF-1? accumulation was reduced by 45% in the presence of NAC indicating that the peak of ROS was only partially involved in its stabilization. On the contrary, NAC did not affect the increase in cell migration in ECs chronically deprived of NO. At variance with acute treatment, chronic L-NAME exposure gave rise to an antioxidant environment characterized by a reduction in cellular ROS content accompanied by an increase in superoxide dismutase-2 (SOD-2) expression and activity. Importantly, this protective response was accompanied by the nuclear accumulation of the transcription factor NF-E2-related factor-2 (Nrf2) that was fully prevented in the presence of NAC. These results suggest the establishment of an antioxidant status in HUVECs chronically deprived of NO in the attempt to neutralize any further cell damage induced by loss of NO. In addition, since NO plays an important role in promoting mitochondrial biogenesis in different cell types and tissues, we analyzed the mitochondrial mass and function in HUVECs after NO deprivation. Long term L-NAME treatment induced a significant reduction in mitochondrial DNA (mtDNA) accompanied by decreases in the incorporation of the metabolic indicator MTS, in cellular ATP content, and in oxygen consumption. In agreement, the silencing of eNOS was able to decrease mtDNA and total cellular ATP levels thus confirming that loss of NO sustained the onset of mitochondrial dysfunction in HUVECs. Importantly, metabolic effects observed in chronically NO deprived ECs was independent of both HIF-1? activity and ROS generation. In conclusion, we demonstrated that an endothelial deficit of NO, by mimicking the in vivo early phase of ED, induces important physiological modifications in human ECs. In particular, loss of NO leads to the accumulation and transcriptional activation of HIF-1? responsible for the enhanced VEGF/KDR expression and cell motility, and to the establishment of mitochondrial dysfunction. Importantly, most of the peculiar features shown by long term NO deprived HUVECs are independent of acute ROS generation, and must therefore depend on other pathways triggered by NO loss. On the contrary, ROS formation appears to be totally responsible for the Nrf2 accumulation that might account for the establishment of an adaptive antioxidant status in response to oxidative stress. Further experiments will be necessary to fully characterize our in vitro model of ED and to elucidate the molecular mechanism(s) involved in HIF-1? stabilization. Our model should however represents an useful system for the study and the identification of innovative pharmacological targets and markers for ED, thus contributing to a better knowledge of the endothelium behavior in the absence of NO and to an improved comprehension of the molecular mechanisms involved in the onset of cardiovascular pathologies.