to stimulate superoxide production; and it causes acute organ injury mainly in the lungs [23]. Over a 10-day period following the administration of paraquat, the death rate even in the VASH1 (+/ 2) KO mice was higher than that in the WT mice; and the survival curves for the WT and VASH1 (+/2) KO mice were thus significantly different (Fig. 7A). Lungs were obtained 48 hours after the paraquat administration and histological analysis was performed. Cellular infiltration, and interstitial edema were evident in VASH1 (+/2) KO mice (Fig. 7B, on the left). Immunohistochemistry for 8-OHdG revealed that DNA damage in VASH1 (+/2) KO (Fig. 7B, on the right). Moreover, protein levels and cell counts in the bronchoalveolar lavage fluid (BALF) were significantly increased in VASH1 (+/2) KO mice (Fig. 7C). Staining for b-gal activity indicated that the intratracheally administered AdLacZ had been delivered to entire bronchial epithelium and some vessels (Figure S3 A and B). When AdVASH1 was intratracheally administered, VASH1 protein synthesis was detected in the lungs for at least 10 days (Figure VASH1 Enhances Stress Resistance of Endothelium S3C). We then tested whether the intratracheal administration of AdVASH1 could protect mice from the paraquat-induced lung injury. Over the same 10-day period, the deaths in the group of VASH1 (+/2) KO mice administered AdVASH1 were signifi- cantly fewer than in the group administered AdLacZ (Fig. 7D). Histological analysis of the lungs revealed that the AdVASH1 treatment attenuated the acute lung injury and DNA damage in VASH1 (+/2) KO mice given AdVASH1 compared with that in those administered AdLacZ (Fig. 7E). Protein levels and cell counts in the BALF were significantly less in the former than in the latter (Fig. 7F). To verify the involvement of SOD2 and SIRT1 in the effect of VASH1 in vivo, we investigated those proteins in the lung tissue, and found that AdVASH1 increased SOD2 and SIRT1 contents in the lungs (Fig. 7G). Since the intratracheal adminis- tration of adenovirus vector tranfered gene mainly in bronchial epithelium, we assumed that VASH1 synthesized by bronchial epithelium affect on neighboring ECs. However, VASH1 might affect the bronchial epithelium as well. NHBECs did not express Figure 6. VASH1 controls SIRT1 level and increases stress resistance of HUVECs. (A) HUVECs were transfected with VASH1 siRNA or control siRNA. After a 72-hour incubation, Western blotting for VASH1 and SIRT1 was performed. (B) HUVECs were transfected with VASH1 siRNA or control siRNA. After a 24-hour incubation, SIRT1 activity was determined (*P,0.01, N = 3) (C) HUVECs were preteated with vehicle or 5 mmol/L SIRT1 activator 3 for 12 hours, and then transfected with VASH1 siRNA or control siRNA. After a 6-day incubation, SA b-gal staining was performed. Scale bars are 100 mm. SA b-gal-positive HUVECs were quantified, and the % of senescent cells was calculated (*P,0.01, N = 4). (D) HUVECs were transfected with SIRT1 siRNA or control siRNA. After a 72-hour incubation, Western blotting for VASH1 and SIRT1 was performed. (E) HUVECs were infected with AdVASH1 or AdLacZ. After a 72-hour incubation, Western blotting for VASH1 and SIRT1. (F) HUVECs were infected with AdVASH1. After a 24-hour incubation, HUVECs were then transfected with SIRT1 siRNA or control siRNA. After a subsequent 24-hour incubation, the cells were exposed to 100 mmol/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 SIRT1 were then performed. 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.g006 PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e46459