Moreover, when ROS were quenched by NAC, premature senescence was partly but significantly inhibited in HUVECs lacking VASH1 (Fig. 5C). Alternatively, overexpression of VASH1 decreased the ROS level when exposed to cellular stresses, and up- regulated SOD2 in HUVECs (Fig. 5D and E). However, when this increase of SOD2 was knocked-down by siRNA, the protective effect of VASH1 on premature senescence was significantly abrogated (Fig. 5F). NAC partly inhibited premature senescence (Fig. 5B). Thus we reasoned that some additional mechanism might be involved. In addition to antioxidants, attention has been recently paid to the protective protein named SIRT1 [21]. Moreover, the synthesis and function of SIRT1 are related to HuR and ATM [22]. Therefore we tested the SIRT1 protein level. The knockdown of VASH1 significantly decreased the level of SIRT1 protein and its activity as well (Fig. 6A and B). However, if this reduced SIRT1 VASH1 Enhances Stress Resistance of Endothelium activity was enhanced by SIRT1 activator 3, the premature senescence of HUVECs lacking VASH1 was notably suppressed (Fig. 6C). Interestingly, the knock-down of SIRT1 increased the VASH1 protein level (Fig. 6D). Alternatively, when VASH1 was overexpressed in HUVECs by AdVASH1, SIRT1 protein significantly increased (Fig. 6E). Moreover, when SIRT1 was knocked-down, the VASH1-mediated protection against stress- induced premature senescence and cell death vanished (Fig. 6E and F). Collectively, the above data showed VASH1 to protect ECs via the induction of SOD2 and SIRT1. VASH1 protects mice from death with acute lung injury induced by paraquat treatment To prove the protective role of VASH1 in vivo, we applied paraquat intoxication to mice. Paraquat is used as a redox cycler Figure 5. VASH1 controls SOD2 level and decreases ROS level in HUVECs. (A) HUVECs were transfected with VASH1 siRNA or control siRNA. After a 24-hour incubation, cellular ROS was determined as described in Materials and Methods (*P,0.01, N = 3). (B) HUVECs were transfected with VASH1 siRNA or control siRNA. Twenty-four hours later, RT-PCR for the indicated genes was performed. (C) HUVECs were transfected with VASH1 siRNA or control siRNA in the presence or absence of 50 mmol/L NAC. After a 12-hour incubation, the culture medium was replaced with growth medium containing vehicle or 50 mmol/L NAC. After a 6-day culture, SA b-gal staining was performed. Scale bars are 250 mm. SA b-gal-positive HUVECs were quantified, and the % of senescent cells was calculated (*P,0.01, N = 3). (D) HUVECs were infected with AdVASH1 or AdLacZ. After a 24- hour incubation, the cells were exposed to 100 mmol/L H2O2 for 1 hour or to 0% FCS/aMEM for 6 hours. Thereafter, the cellular ROS level was determined (*P,0.01, N = 3). (E) HUVECs were infected with AdVASH1 or AdLacZ. After a 24-hour incubation, RT-PCR for VASH1 and SOD2 was performed. (F) HUVECs were infected with AdVASH1 or AdLacZ. After a 24-hour incubation, HUVECs were then transfected with SOD2 siRNA or control siRNA. After a subsequent 24-hour incubation, the cells were exposed to 100 m mol/L H2O2 for 1 hour followed by a 48-hour incubation in growth medium. Scale bars are 250 mm. SA b-gal staining and Western blotting for VASH1 and SOD2 were then performed. SA b-gal-positive HUVECs were quantified, and the % of senescent cells was calculated (*P,0.01, N = 3). All the studies were repeated at least 3 times to confirm the reproducibility. doi:10.1371/journal.pone.0046459.g005 PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e46459