killed by those stresses (Fig. 2C). In contrast, HUVECs overex- pressing VASH1 were resistant to those cellular stresses (Fig. 2D). Importantly, stress-induced HUVEC premature senescence could be prevented by medium conditioned by stable VASH1 transfectant (Fig. 2E). We applied HAECs as another primary ECs. Identical to HUVECs, HAECs became senescent when VASH1 was knocked- down (Fig. 3A and B). Alternatively, HAECs became resistant to cellular stresses when VASH1 was overexpressed (Fig. 3C and D). We previously established stable human VASH1 transfectants of MS1 [8]. Those stable transfectants were also resistant to cellular stresses (Figure S1 A and B). Collectively, the above data showed VASH1 to protect ECs from premature senescence and to make them resistant to stress- induced cell death. VASH1 protein level is determined via HuR-mediated posttranscriptional regulation We examined whether cellular stress modulated the expression of VASH1 in ECs. We observed that cellular stress increased VASH1 protein level at 1–6 hour time points without the increase of VASH1 mRNA (Fig. 4A), indicating that post-transcriptional regulation might operate to increase the level of VASH1 protein when ECs were exposed to stress. Hu proteins are RNA-binding VASH1 Enhances Stress Resistance of Endothelium proteins that bind to AU-rich elements (AREs) in the 39 untranslated region (UTR) of mRNAs [17]. We could show such elements in the 39 UTR of VASH1 (Fig. 4B). Among Hu proteins, HuR is mostly related to cellular stress responses [18]. ChIP assay proved that HuR protein bound to this region in HUVECs (Fig. 4C). Furthermore, the knock-down of HuR abrogated the increase in VASH1 protein upon cellular stress in HUVECs (Fig. 4D). These results suggest that VASH1 in the ECs was targeted by HuR. VASH1 protects ECs via the induction of SOD2 and SIRT1 VEGF produced by ECs is reported to be a survival factor for ECs themselves [19]. SU5416, a VEGF receptor kinase inhibitor, did not affect the basal expression of VASH1 in HUVECs, but induced EC death (Figure S2 A and B). This EC death could be diminished by AdVASH1, but the senescence phenotype induced by VASH1 siRNA could not be reversed by exogenous VEGF (Figure S2B and C). These results suggest that the effect of VASH1 may not have involved the VEGF signaling. One of the major causes of stress-induced premature senescence is ROS [20]. We could show that cellular ROS was significantly higher in HUVECs lacking VASH1 (Fig. 5A). We sought the reason for this increase in the ROS level in HUVECs lacking VASH1. Among various antioxidants tested, SOD2 was found to be down-regulated in HUVECs lacking VASH1 (Fig. 5B). Figure 4. HuR increases VASH1 protein level in HUVECs. (A) HUVECs were incubated in 0% FCS/aMEM, and total RNA and protein were extracted at the indicated time points. Thereafter, RT-PCR and Western blotting for VASH1 were performed. Values below each band represent the mean fold change in RNA or protein expression level compared with the cognate zero time. (B) The AU-rich element (ARE) in the 39 non coding region of the VASH1 gene is shown. (C) Immunoprecipitation and reverse transcription-polymerase chain reaction were performed as described in Materials and Methods. (D) HUVECs were transfected with HuR siRNA or control siRNA. After a 24- hour incubation, total RNA and protein were extracted; and then RT-PCR for HuR and Western blotting for VASH1 were performed. All the studies were repeated at least 3 times to confirm the reproducibility. doi:10.1371/journal.pone.0046459.g004 PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46459