NO Deficiency and HIF-1a6 Stabilization in HUVECs Figure 3. Effects of L-NAME treatment on eNOS, VEGF and KDR expression. (A) Densitometric analysis of eNOS protein expression. ***p,0.001; t test; n = 11. Inset: a representative blot out of eleven is shown. Total eNOS protein was evaluated by western blotting on lysates prepared from control cells (lane 1) or from 48 h L-NAME treated cells (lane 2). b-actin was used as a loading control. (B) eNOS RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. No significant differences between control and L-NAME treated cells (t test, n = 3). (C) VEGF and KDR RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. **p,0.01 vs control cells (CTRL); t test; n = 624 for VEGF and KDR, respectively. (D) VEGF protein levels were detected by ELISA measurement in conditioned media collected from control or 48 h L-NAME treated cells. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). **p,0.01; t test; n = 3. (E) KDR protein was visualized by western blot after immunoprecipitation with KDR antibodies of HUVEC lysates obtained from control (lane 1) or from 48 h L-NAME treated cells (lane 2). An aliquot of total cell lysates was immunoblotted with b-actin antibodies as a control (input). Shown is a representative blot of 2 comparable experiments. (F) Control cells (lanes 1 and 2) or 48 h L-NAME treated cells (lanes 3 and 4) were stimulated for 5 min with 25 ng/ml VEGF. Aliquots of cell lysates were separated by 10% SDS-PAGE and immunoblotted with the indicated antibodies. Actin was used as a loading control. Shown is a representative blot of 4 comparable experiments. doi:10.1371/journal.pone.0029680.g003 using RNA interference methodology. HUVEC transfection with eNOS siRNA caused a mean reduction in eNOS protein levels of 7060.1% (Figure 6A). In agreement with the results obtained after long-term exposure to L-NAME, HIF-1a accumulated in the nucleus of eNOS knock-down cells, whereas cells transfected with control siRNA were unaffected (Figure 6B). Moreover, in eNOS silenced HUVECs, again similarly to long term L-NAME treated cells, VEGF production was increased (Figure 6C), while the amount of mtDNA as well as ATP levels were decreased (Figure 6D). Discussion Our results demonstrate that long-term pharmacologic and genetic down-regulation of eNOS induce relevant effects in HUVECs. The long-term treatment with L-NAME induced a significant decrease in mtDNA amount, accompanied by de- creased mitochondrial activity and ATP production. This observation is in agreement with previous results, which showed the important role of the gas in promoting mitochondrial biogenesis in different cell types and tissues [16,17]. Similar results (i.e. decreased mitochondrial mass) were obtained in the microvasculature of mice treated with NG-monomethyl-L-arginine (L-NMMA) for a long period of time (1–2 months) [24]; however, given the length of the treatment, these results are not directly comparable to ours. To our knowledge, the effect of NO deprivation for shorter times (days) on mitochondrial biogenesis in ECs had not previously investigated. Our work demonstrated that HUVECs respond to NO deprivation already after 48 h of treatment, making them a useful model for the investigation of the effects of the gas on mitochondrial biogenesis and function. An important consequence of the decrease in NO production observed by us is the accumulation of HIF-1a in the nucleus, with a resulting adaptation of gene expression to a pseudohypoxic state, PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29680