results suggest that basal NO, at variance with the gas released acutely in response to motogenic factors, diminishes the migratory ability of ECs. The tonic inhibitory effect of basal NO on migration, by acting as a brake on inappropriate migration, could prevent exaggerated angiogenic responses and thus be an important homeostatic factor in EC physiology. In the present study, we have further investigated the effects of chronic NO deprivation on EC physiology, and attempted to unravel the pathway linking basal NO to migratory ability. Results obtained both by long term pharmacological inhibition and by genetic silencing of eNOS indicate that NO loss induces profound modifications in EC physiology, leading to a general decrease of mitochondrial mass and metabolic activity, to an accumulation of Hypoxia Inducible Factor-1a (HIF-1a) in normoxia and to enhanced chemotactic migration as a consequence of the increased HIF-1a levels. These results have important implication for our understanding of the consequences of NO deprivation in cardiovascular pathology. Results HUVECs chronically treated with L-NAME are not apoptotic, but have decreased mitochondrial mass and function To characterize the effects of long term NO deprivation on human ECs, we first analyzed possible changes in cell viability. As shown in Figure 1A, treatment with L-NAME for 48 h did not induce caspase-3 cleavage, which instead occurred when HU- VECs were exposed to high glucose (30 mM for 48 h), a condition known to be apoptotic for these cells [15]. Moreover, quantifica- tion of apoptosis/necrosis by annexin V-conjugated FITC and PI staining followed by FACS analysis did not show any difference in the apoptotic index between control and L-NAME treated HUVECs (0.1660.03 and 0.1560.05 in control and L-NAME treated cells, respectively). Also the percentage of necrotic cells was unaffected by the treatment, ranging from 8.360.26% in control cells to 4.160.21% in cells treated with L-NAME. Finally, we checked the levels of Bcl-2 and Bax, well-known proteins involved in the regulation of apoptosis endowed with anti-apoptotic and pro-apoptotic activity respectively, and found that their expression was unchanged by L-NAME treatment (Figure 1B). In HeLa cells and adipocytes, basal NO enhances mitochon- drial mass [16,17], however the effect of the gas on mitochondrial biogenesis in ECs had not been investigated. As shown in Figure 1C, HUVECs treated with L-NAME for 48 h showed a decreased amount of mitochondrial DNA (mtDNA) as compared to untreated cells (by 3860.05%). Moreover, incorporation of the metabolic indicator MTS was lower in L-NAME treated cells (with a mean reduction of 2665%) (Figure 1D) indicating a decreased mitochondrial activity in NO deficient cells as compared to control cells. In agreement, oxygen consumption as well as ATP levels were reduced in the L-NAME treated cells (Figures 1E and F). The NO donor DETA-NO completely reverts the increased migratory behavior induced in HUVECs by long-term L- NAME treatment Our previous results demonstrated that long-term treatment of HUVECs with L-NAME induced a strong increase of the cell migratory capacity assayed in Boyden chambers [14]. These results suggested that chronic, constitutive NO production exerts a tonic inhibition on HUVEC migratory behavior. To validate this hypothesis, we investigated whether long-term administration of the slow NO donor DETA-NO would reverse the L-NAME- NO Deficiency and HIF-1a6 Stabilization in HUVECs enhanced migration of HUVECs. As shown in Figure 2A, chronic treatment of HUVECs with L-NAME for 48 h was confirmed to increase the cell migratory response to VEGF by about 45%. Notably, also basal migration was augmented in a similar manner. No significant increase in cell migratory behavior was observed after 24 h treatment with L-NAME (data not shown). A dose as low as 500 nM of DETA-NO, supplied to the L- NAME treated cells for the last 24 h of the treatment, completely abolished the increased migration observed in both basal and VEGF-stimulated NO deficient HUVECs, thus confirming that enhanced HUVEC migration caused by L-NAME was due to NO deprivation. Notably, cells treated with the combination of L- NAME and DETA-NO showed diminished migration compared to cells treated with the donor alone. This result can probably be explained by a biphasic effect of chronic NO, as demonstrated for HeLa cells in our previous work [14]. To investigate whether the increased migratory behavior caused by chronic NOS inhibition was due to a deficiency in cyclic GMP (cGMP) consequent to NO deprivation, we evaluated the effect of long-term treatment with the guanylate cyclase inhibitor ODQ on HUVEC migration in Boyden chambers. After treatment of the cells for 48 h either with L-NAME or with ODQ, migration assays were performed in the absence of the drugs. As shown in Figure 2B, chronic treatment with ODQ did not induce any change either in basal or in VEGF-stimulated HUVEC motility. The measurement of cGMP accumulation in HUVECs chroni- cally treated with L-NAME or ODQ confirmed the ability of both drugs to significantly reduce cGMP levels (Figure 2C). These results indicated that blunting of the cGMP signaling pathway is not involved in the stimulatory effect induced by NO depletion. Effects of long term L-NAME treatment on eNOS, VEGF, KDR expression and VEGF signaling in HUVECs We previously considered the hypothesis that the improved migratory ability of HUVECs chronically treated with L-NAME could be caused by a rebound effect: expression of eNOS in response to its chronic inhibition could have been increased, thus stimulating migration via enhanced NO release upon exposure of the cells to motogenic factors in the absence of the inhibitor. Our analysis, however, suggested that eNOS was not increased by chronic L-NAME treatment [14]. To investigate this phenomenon further, we quantitatively analyzed both eNOS protein and mRNA levels in treated and untreated cells. As shown in Figure 3A, a significant decrease in eNOS protein levels (by 4865%) was detected in treated cells. In contrast, no variation in the eNOS mRNA level, measured by RT-qPCR, was observed (1.0460.3 fold in comparison to untreated cells) (Figure 3B), suggesting that chronic inhibition of eNOS causes an increased degradation of the enzyme and/or impairment of the translation of its mRNA. In an attempt to explain the mechanism through which NO deprivation enhances migration, we investigated how chronic L- NAME treatment affects the expression levels of the VEGF receptor-2 (kinase insert domain receptor, KDR). We also analyzed VEGF itself, as endogenous production of the growth factor could potentiate migration by an autocrine loop. RT-qPCR analysis demonstrated that both VEGF and KDR mRNA levels increased, 1.9160.2 and 1.7960.2 fold respectively, in treated compared to untreated cells (Figure 3C). In addition, increased VEGF production and KDR protein expression was demonstrated by ELISA measurement and biochemical analysis of HUVEC lysates, respectively. As shown in Figure 3D, quantitative measurements of the secreted protein revealed a 1.7-fold increase of VEGF in conditioned media from L-NAME treated cells in PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29680