AMPK-Dependent Angiogenesis by Nitrated Lipids Figure 5. HIF-1a mediates OA-NO2-induced intracellular Ca2+ accumulation and AMPK activation. A) BAECs were treated with YC-1 (30 mM) for 30 min followed by incubation with OA-NO2 for the indicated times. AMPK phosphorylation and protein levels were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. B) BAECs were incubated with HIF- 1a-specific siRNA or control siRNA for 48 h and then treated with OA-NO2 or vehicle for 16 h. After treatment, cell lysates were analyzed for HIF-1a and AMPK protein levels and AMPK phosphorylation at Thr172. Lower panels: summary data (*p,0.05 vs. control; #p,0.05 vs. OA-NO2 group; n = 3). C) BAECs were incubated with OA-NO2 (2.5 mM) for the indicated times. Intracellular Ca2+ was measured with Fluo-4 fluorescent dye as described in Materials and Methods. *p,0.01 vs. control (n = 4). D) BAECs were pre-treated with YC-1 (30 mM) or BAPT-AM (25 mM) for 30 min or HIF-1a siRNA or control siRNA for 48 h followed by incubation with OA-NO2 for 16 h. After treatment, intracellular Ca2+ was measured in intact cells using a Fluo-4 NW kit. *p,0.05 vs. control; #p,0.05 vs. OA-NO2 group (n = 3). E) BAECs were pre-loaded with 25 mM BAPT-AM for 30 min prior to incubation with 2.5 mM OA-NO2 for 16 h. AMPK protein levels and phosphorylation at Thr172 were detected as described above. Representative blots (top) and densitometric analyses (bottom) are shown. Values are means 6 SD from three independent measurements. *p,0.05 vs. control; #p,0.05 vs. OA- NO2 group. doi:10.1371/journal.pone.0031056.g005 Next, we determined whether OA-NO2-induced eNOS phos- phorylation was associated with increased NO release. Treatment with OA-NO2 significantly increased NO release, an effect that was inhibited by Ad-DN-AMPK transfection (Figure 7D). Impor- tantly, Ad-DN-AMPK transfection reduced AMPK activity in OA-NO2-treated cells to below control levels (Figure 7E). These results indicate that OA-NO2 increases NO release via AMPK activation. Discussion OA-UFAs are unique signaling mediators that are present in a variety of cell types, including endothelial cells [29]. Considerable evidence points to a role for circulating OA-UFAs in vascular- protective effects [9], although the mechanisms by which OA-NO2 are incompletely understood. In this study, we report that OA- NO2 activates AMPK in endothelial cells via a Ca2+-dependent pathway, and we implicate CaMKKb as the responsible upstream kinase. Our results show that HIF-1a is an inducer of the intracellular Ca2+ mobilization that leads to AMPK activation. On the basis of the results of experiments with an HO-inhibitor and HO-1 siRNA, we also implicate HO-1 in OA-NO2-induced upregulation of endothelial HIF-1a. HO is a rate-limiting enzyme in heme degradation and functions to convert heme to biliverdin, carbon monoxide (CO) and iron. Human HO occurs in two main isoforms, the inducible HO-1 form and the constitutive HO-2 form. Previous studies demonstrated that HO-1 exerts anti-inflammatory effects [30], including prolongation of cardiac xenograft graft survival [31] and inhibition of leukocyte transendothelial migration during comple- ment-dependent inflammation [32] and in response to low-density lipoprotein (LDL) oxidation [33]. More importantly, once induced, HO-1 also confers vascular cytoprotection [30]. The importance of this result is demonstrated by the severe and persistent endothelial damage observed in the case of human HO- 1 deficiency [34] and in gene-targeted mice deficient in HO-1 [35]. Furthermore, induction of HO-1 may directly regulate endothelial cell activation, preventing adhesion molecule expres- sion and chronic graft rejection [36]. In vitro, HO-1 protects endothelial cells from hydrogen peroxide–mediated cell death [37] and from tumor necrosis factor a (TNFa) cytotoxicity [38]. In addition, HO-1 has been suggested to play a role in angiogenesis. PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31056