endogenous VASH1 (Figure S4A). In addition, the overexpression of VASH1 in NHBECs did not alter the level of SOD2 and SIRT1 (Figure S4B) nor the cell death after the treatment with H2O2 (Figure S4C). We therefore judge that the protective action of VASH1 in the paraquat-induced acute lung injury occurred mainly through ECs. Discussion Our previous studies on VASH1 were focused on the inhibition of angiogenesis. Here we noticed, to our surprise, that the knock- down of basal VASH1 expression resulted in the premature senescence of ECs. We extended our study and revealed for the first time that VASH1 protected ECs from premature senescence and cell death when the cells were exposed to oxidative or serum- starvation stress. Angiogenesis inhibition generally causes vascular regression by inducing EC death, which regression may also result in proteinuria and hypertension in vivo [24]. We noted earlier that VASH1 neither instigates such vascular regression nor causes proteinuria and hypertension [25,26]. Our present results further highlight the uniqueness of VASH1 in that it not only inhibited angiogenesis but also enhanced the maintenance of ECs by strengthening resistance against stress. The presice mechanism of the action of VASH1 remains to be elucidated. VASH1 mRNA is induced by stimulation with angiogenic factors such as VEGF and FGF-2 via the activation of PKC-d [27]. Here we observed that the VASH1 protein level increased without the increase in VASH1 mRNA when ECs were exposed to cellular stress, suggesting the posttranscriptional gene regula- tion. One of the important mechanisms of posttranscriptional regulation is the rapid degradation of mRNAs signaled by AREs in their 39 UTR. The Hu family of RNA-binding proteins binds to AREs in the 39UTRs of the target mRNAs, prevents their degradation, and enhances their translation [18]. There are 4 members of Hu proteins; HuB, HuC, HuD and HuR. Whereas HuB, HuC, and HuD are selectively expressed in the nervous system and play roles in neuronal differentiation and plasticity, HuR is ubiquitously expressed and exhibits numerous functions mostly related to cellular stress responses [18]. Thus, we consider the stress-induced VASH1 protein synthesis to have been regulated by HurR. Here we gave evidence for 2 proteins as targets of VASH1 for the maintenance of ECs, the first being SOD2. The SOD family forms the major antioxidant defense system, which consists of 3 members: SOD1 as the cytoplasmic Cu/Zn-SOD, SOD2 as the mitochondrial Mn-SOD, and SOD3 as the extracellular Cu/Zn- SOD [28]. Because of its localization in mitochondria, SOD2 is the first line of defense against oxidative stress [29]. ECs are known to express a high level of SOD2 [30], and SOD2 is thought to play a principal role in protecting the vascular system from oxidative stress generated by various pathophysiological processes [31]. The second target of VASH1 we discovered was SIRT1. SIRT1 is a member of mammalian NAD+-dependent deacetylase family. Among them, SIRT1 is widely expressed, and is now considered to be responsible for the protection of cells from various types of stress [32]. Particularly, a number of reports indicate that vascular SIRT1 protects vessels from various vascular diseases including atherosclerosis and diabetic vascular complications [33– 36]. The knock-down of VASH1 decreased the expression of SIRT1, whereas the knock-down of SIRT1 increased the expression of VASH1 in ECs. This may suggest that VASH1 lays upstream of SIRT1 in the axis of VASH1-SIRT1 in ECs. SIRT1 is expressed in ECs during angiogenesis [37]. The VASH1 Enhances Stress Resistance of Endothelium correlation of VASH1 and SIRT1 in the regulation of angiogen- esis needs to be determined in future. As mentioned earlier, angiogenesis inhibitors induce EC death and vascular regression [24]. Hence, the most intriguing aspect of VASH1 is simultaneous angiogenesis inhibition and EC protec- tion. It is well documented that inflammatory cells form dense infiltrates at the site of angiogenesis and that oxidative stress is the major characteristic of such inflammatory conditions [38,39]. Moreover, ROS can be one of the mediators of angiogenesis as well [27]. For that reason, we propose that the function of VASH1 is to halt angiogenesis and stabilize neo-vessels. VASH1 is highly expressed in ECs at sites of angiogenesis. However, besides its presence there, we noticed previously that VASH1 protein is detectable in arterial ECs under the basal condition [40]. Arterial ECs are exposed to various physical forces. Moreover, oxidative stress-induced DNA damage is thought to play an important role in vascular senescence and senescence- related vascular diseases [41]. We therefore suggest that such VASH1 in the arterial wall is available there for the protection of vessels. Indeed we noted in earlier studies that VASH1 can prevent intimal thickening of arteries as well as diabetic renal injury [12,42]. The lungs are the organ with the highest exposure to ambient air among all of the organs in the body. Because of its large alveolus surface and affluent blood perfusion, the lung tissue is most susceptible to oxidative injury. Here we used Paraquat to induce acute lung injury and showed that the intrabronchial sdministration of AdVASH1 protected lungs from acute lung injury. Since the intratracheal administration of adenovirus vector tranfered gene mainly in bronchial epithelium, we assumed that VASH1 synthesized by bronchial epithelium should affect on neighboring ECs in a paracrine manner. The excessive oxidative stress is thought to be one of the major causes of various lung diseases including chronic obstructive pulmonary diseases (COPD), pulmonary hypertension, and the post-reperfusion injury of transplanted lungs [43–45]. Moreover, there are several reports describing the relationship among SOD2, SIRT1, and COPD [46–48]. It would be therefore interesting to see if there is any relationship between those pulmonary diseases and VASH1. In summary, our present study revealed that VASH1 not only inhibited angiogenesis but also enhanced the maintenance of ECs by strengthening their resistance against stress. We showed SOD2 and SIRT1 to be targets of VASH1 in ECs for strengthening this resistance. The close relationship among VASH1, SOD2 and SIRT1 may indicate the protective value of VASH1 in the vascular system. Supporting Information Figure S1 VASH1 over-expression inhibits the cell death of MS1 cells induced by cellular stresses. Stable VASH1- expressing MS1 clones and mock MS1 transfectants were exposed to 200 mmol/L H2O2 (A) or serum starved (0% FCS/DMEM, B) for 24 hours. The trypan blue exclusion assay was then performed to judge cell death. Blue-stained cells were quantified, after which the % of dead cells was calculated. Values are the ratio of blue-stained cells to total cells, and are the means and SDs of 3 wells. *Significant difference compared with the value for the corresponding mock. (TIFF) Figure S2 VASH1 protects HUVEC death by the treat- ment with a VEGF receptor inhibitor. (A) HUVECs were incubated in growth medium containing SU5416. Total RNA and protein were extracted from the cells at the indicated times, and then RT-PCR and Western blotting for VASH1 were performed. PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e46459