This supposition is supported by the observation that overex- pression of HO-1 induces proliferation and formation of capillary-like structures [39]. Thus, therapeutic induction of HO-1 may be beneficial in the treatment of chronic inflamma- tory diseases as well as cardiovascular diseases. Our results indicate that OA-NO2 potently induced HO-1 protein expression in endothelial cells. In agreement with our studies, other studies have suggested that HO-1 expression is induced in human aortic endothelial cells (HAECs) by LNO2, which shows a higher potency in this context than other established stimuli, including oxidized fatty acids and hemin. This induction of HO-1 expression by NO2-FA is not mediated by NO, NF-kB, or PPARc [40]. A recent study conducted by Liu et al. [41] suggested that AMPK activation regulates HO-1 gene expression in endothelial cells via the Nrf2/antioxidant responsive element signaling pathway and that HO-1 contributes to the biological actions of this kinase. Our data do not exclude the possibility that AMPK activation upregulates HO-1 expression at the gene transcription level. As shown in Figure 1E, Ad-DN-AMPK partially blocks OA-NO2-induced HO-1 protein levels, suggest- ing that AMPK activation may play a small role in HO-1 protein expression; however, OA-NO2 induces HO-1 expression 2.5-fold as early as 4 h after incubation when AMPK is not yet activated (Figure 1B), implying that OA-NO2 induces HO-1 directly or via another unknown pathway. Thus, AMPK does not act upstream of HO-1 production in this system. Current data supports the idea that upregulation of human HO-1 expression by NO2-FA requires synergy between the cAMP-dependent response element and the AP-1 sequences in the 24.5 kb HO-1 promoter region [6,40]; however, the mechanisms involved in these events remain poorly characterized, and the identities of the signaling molecules downstream of HO-1 that mediate the vascular protective effects of nitrated lipids are not entirely known. The novel finding of this study is that OA-NO2 induces HIF- 1a expression via HO-1 under non-hypoxic conditions. HIF-1, which exists as a heterodimer composed of the HIF-1a and HIF- 1b subunits, has been shown to mediate numerous physiological and pathophysiological responses to hypoxia. Under normoxic conditions, however, HIF-1a is ubiquitinated and rapidly degraded [42] and is thus present at very low levels under these conditions. Under hypoxic conditions, the HIF-1a subunit is induced. Because HIF-1b is constitutively expressed, HIF-1a is responsible for conferring hypoxia sensitivity to heterodimeric HIF-1. Using specific chemical inhibitors, we demonstrated that ERK1/2 partially mediated HO-1–induced HIF-1a expression (Figure 4). In addition to inducing HIF-1a expression, ERK1/2 has been reported to phosphorylate HIF-1a and to thereby increase its activity [20]. The possibility that CO, a metabolite of HO-1 [43], also plays a role in HIF-1a induction by HO-1 cannot be excluded by our data. A recent study by Chin et al. [44] suggested that exposure of macrophages to CO resulted in rapid HIF-1a activation and stabilization, which regulates the expression of genes involved in inflammation, metabolism, and cell survival. This previous study also provided evidence that CO may serve as a signaling intermediary between HO-1 and HIF-1a. Another important find of the present study is that HIF-1a contributes to an increase in [Ca2+] , which is responsible for i CaMKKb-dependent AMPK activation (Figure 5 and 6). Pharmaceutical and genetic inhibitors of HIF-1a suppressed OA-NO2-induced increases in [Ca2+]i, suggesting that HIF-1a is involved in the OA-NO2-induced mobilization of Ca2+ in endothelial cells. Previous studies demonstrated that hypoxia AMPK-Dependent Angiogenesis by Nitrated Lipids evokes an increase in [Ca2+]i in endothelial cells [45], and presumably, the induction of HIF-1a contributes to this increase in [Ca2+]i under hypoxic conditions or under normoxic conditions in the presence of induction factors that activate HIF-1a. The mechanisms that are involved in endothelial Ca2+ homeostasis within the vasculature following HIF-1a activation are also largely unknown. Although HIF-1a has been reported to regulate Ca2+ homeostasis in pulmonary arterial smooth muscle cells via upregulation of store-operated Ca2+ channels and enhanced Ca2+ influx [24], the detailed mechanisms remain to be elucidated. Our results also showed that OA-NO2-stimulated AMPK activation was inhibited by chelation of intracellular free Ca2+, selective inhibition of CaMKK by STO-609, selective inhibition of Ca2+/calmodulin-dependent protein kinase by KN-93, and siRNA-mediated silencing of CaMKK-b expression (Figure 6). In addition, the association between AMPK and CaMKK was enhanced by OA-NO2 treatment. Taken together, these data indicate that the HIF-1a/Ca2+/CaMKK- pathway is crucial for OA-NO2-induced AMPK activation. Furthermore, CaMKK- mediated AMPK activation in endothelium has also recently been reported in response to thrombin [46], adenosine diphosphate (ADP) [47], and bradykinin [48]. Results from previous studies indicated that endothelial AMPK may play an important physiological role in the function of both endothelial cells and the cardiovascular system as a whole; thus, activation of AMPK may provide an explanation for the beneficial effects of OA-NO2 on these systems (see review [49]). Our findings show that induction of the CaMKKb/ AMPK pathway by OA-NO2 in endothelial cells may have both physiological and therapeutic relevance. First, endothelial AMPK activation by OA-NO2 activates nitric oxide synthase (via phosphorylation on Ser 1177) and elevates NO bioavail- ability, and these effects may not only protect against early events in atherogenesis, such as white cell adherence [50], but may also prevent later steps in atherogenesis, including fibrous plaque formation. Endothelial NO likely represents the most important anti-atherogenic defense molecule in the vasculature [50]. Nitrated lipids act as NO donors in vitro and are widely considered to be a possible endogenous source of NO [51]. Enhanced NO production under hyperlipidemic and hypercho- lesterolemic conditions, such as those that occur with obesity or insulin-resistant status, however, has been associated with low NO bioactivity [52]. Therefore, it is reasonable to postulate that the presence of these nitrated products and the related AMPK/ eNOS/NO pathway in vivo may actually play a compensatory role, providing an adjustable supply of NO to compensate for the impaired NO bioactivity and endothelial-dependent vasorelax- ation that is characteristic of the early steps of vascular disease. Second, AMPK signaling acts as a novel regulator of angiogen- esis and is specifically required for endothelial cell migration and differentiation under conditions of hypoxia [53] or in response to adiponectin [54]. Additionally, AMPK-dependent eNOS activity is required for adequate endothelial tube formation [54]. As described above, increased levels of nitrated lipids are formed in the context of the hyperlipidemia associated with obesity and insulin resistance. Thus, the induction of AMPK and angiogen- esis by nitrated lipids may also represent an adaptive defense mechanism against impaired angiogenesis and/or vascular injury caused by obesity-related dislipidemia. The role that activated AMPK plays in increasing fatty acid oxidation via phosphory- lation and inhibition of ACC and leading to a decrease in the concentration of malonyl-CoA is most important [55]. In PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31056