Determination of protein and cell counts in the broncheoalveolar lavage fluid (BALF). Broncheoalveolar lavage was performed by intratracheal injection of PBS (0.7 ml) for 2 times. BALF was collected and centrifuged at 400 g for 5 min at 4uC. The recovered fluid was processed for determination of protein concentration (DC Protein Assay; Bio-Rad Laboratories, Inc, CA). The pelleted cells were resuspended in 1 ml PBS and counted. Calculations and statistical analysis Data were expressed as means 6 SDs. The statistical significance of differences between groups was evaluated by use of the unpaired ANOVA, and P values were calculated by performing the unpaired Student’s t test. The significance between survival curves was analyzed by Kaplan-Meier survival analysis with log-rank testing. A value of P,0.05 was the criterion for significance. Results VASH1 protects ECs from premature senescence and stress-induced cell death To understand the function of VASH1, we applied siRNA- mediated knock-down of VASH1 expression in HUVECs (Fig. 1A). We noticed that HUVECs lacking VASH1 became flatter under the basal condition (Fig. 1B). As this phenotype VASH1 Enhances Stress Resistance of Endothelium resembles that of senescent cells, we assessed the cellular senescence. There was a significant increase in SA b-gal reactivity in HUVECs lacking VASH1 (Fig. 1B). As ATM is known to be phosphorylated during premature senescence of ECs [14], we examined the level of phosphorylated ATM (p-ATM) in HUVECs lacking VASH1 and found an increase in it (Fig. 1C). It was described that autophagy is associated with premature senescence [15]. Immunostaining for LC3, a marker of autophagy, revealed autophagy in HUVECs lacking VASH1 (Fig. 1D). These results indicate that the knock-down of VASH1 caused the premature senescence of ECs. Although VASH1 lacks a classical signal sequence, it is secreted by binding to the small vasohibin-binding protein [16]. Accordingly, treatment of HUVECs with blocking monoclonal anti-VASH antibody induced a similar senescence phenotype (Fig. 1E), suggesting the importance of secreted VASH1 in protecting against cellular senescence. We next overexpressed the human VASH1 gene in HUVECs (Fig. 2A). Basal expression of VASH1 in ECs varies depending on the culture condition, as sparse HUVECs express less whereas subconflunet to confluent HUVECs express more VASH1 [3]. We therefore used sparse HUVECs for the overexpression. When those HUVECs were exposed to H2O2 or serum starvation, the AdVASH1-infected HUVECs exhibited resistance to premature senescence (Fig. 2B). We examined cell death after the exposure to cellular stresses. When HUVECs lacking VASH1 were exposed to H2O2 or serum starvation, they were vulnerable and easier to be Figure 3. VASH1 inhibits premature senescence and cell death of HAECs. (A) HAECs were transfected with VASH1 siRNA or control siRNA. After a 24-hour incubation, RT-PCR and Western blotting for VASH1 were performed. (B) After a 6-day incubation, SA beta-gal staining was performed on HAECs that had been transfected with VASH1 siRNA or control siRNA. SA beta-gal-positive HAECs were quantified, and the % of senescent cells was calculated (*P,0.01, N = 3). (C) HAECs were infected with AdVASH1 or control AdLacZ. After a 24-hour incubation, RT-PCR and Western blotting for VASH1 were then performed. (D) HAECs infected with AdVASH1 or control AdLacZ were exposed to 100 mmol/L H2O2 or to 0% FCS/DMEM for 24 hours. The trypan blue exclusion assay was performed to judge cell death (*P,0.01, N = 3). All the studies were repeated at least 3 times to confirm the reproducibility. doi:10.1371/journal.pone.0046459.g003 PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e46459