AMPK-Dependent Angiogenesis by Nitrated Lipids Figure 1. Induction of HO-1 protein and AMPK activation by OA-NO2. A) BAECs were incubated with OA-NO2 at the indicated concentrations or with BSA (vehicle) for 16 h, and western blot analysis was performed as described in Materials and Methods to detect HO-1 protein expression and AMPK phosphorylation at Thr172. The blot is representative of those obtained from three separate experiments. Corresponding densitometric analyses of phosphorylated AMPK and ACC are shown. *p,0.05 vs. control. B) BAECs were incubated with 2.5 mM OA-NO2 for the indicated times, and western blotting was performed as above. The blot is representative of three blots obtained from three separate experiments. *p,0.05 vs. corresponding control. C) Confluent BAECs were exposed to vehicle or OA-NO2 (2.5 mM) for 16 h. AMPKa was immunoprecipitated from cell lysates (1 mg) with a specific antibody. AMPK activity was assayed by 32P-ATP incorporation into the SAMS peptide. *p,0.05 vs. control. D) BAECs were incubated with the indicated concentrations of OA for 16 h. Western blotting was performed as described in Materials and Methods. E) BAECs were infected with Ad-DN-AMPK (MOI = 50) or Ad-GFP (control). Infected and non-infected cells were treated with 2.5 mM OA-NO2 for 16 h. AICAR and metformin were used as positive controls. The blot is representative of three blots obtained from three separate experiments. doi:10.1371/journal.pone.0031056.g001 associated with ferrous iron metabolism [21]. Given that OA-NO2 induced both HO-1 expression and AMPK activation, we determined whether OA-NO2 also influenced HIF-1 expression. As predicted, OA-NO2 induced an increase in HIF-1a in HUVECs with a time course similar to OA-NO2-induced HO-1 expression and AMPK phosphorylation (Figure 3A). To explore the potential involvement of HO-1 in OA-NO2-induced HIF-1a expression, we pre-treated cells with the selective HO inhibitor zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG) and HO-1-specific siRNA. As shown in Figure 3A and B, ZnBG and HO-1 siRNA treatment abrogated the induction of HIF-1a protein expression and AMPK phosphorylation by OA-NO2, suggesting that OA-NO2 acts through the induction of HO-1 to increase HIF-1a expression and subsequently activate AMPK. The efficacy of the HO-1 siRNA in the downregulation of HO-1 expression was confirmed by western analysis (Figure 3B). ERK1/2 is partially responsible for OA-NO2/HO-1- mediated HIF-1a induction Clearly, the HIF pathway plays an important role in the regulation of metabolism under hypoxic conditions; however, a variety of HIF-1 stimuli function independently of oxygen concentration. These stimuli are primarily mediated by proteins that regulate HIF-1 translation. This pathway contrasts with hypoxic stimuli, which act upon pre-existing a-subunits [20]. HIF- 1a activation by non-hypoxic stimuli has been linked primarily to the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the mitogen-activated protein kinase (MAPK) pathway [22]. Both kinase pathways are known to be intimately associated with the regulation of HIF-1a protein translation, stabilization, and transcriptional activity [23]. Our data indicate that OA-NO2 did not activate PI3-K/Akt (data not shown). To determine whether OA-NO2-mediated HIF-1a induction is dependent on the MAPK pathway, we examined p38 MAPK and extracellular signal- regulated kinase 1/2 (ERK1/2) phosphorylation following stim- ulation with OA-NO2. Treatment with OA-NO2 induced a dose- dependent increase in the phosphorylation of ERK1/2 and p38 MAPK (Figure 4A). ZnBG inhibited OA-NO2-induced activation of ERK1/2, suggesting a role for HO-1 in this activation (Figure 4B). We next determined whether ERK1/2 was a potential upstream mediator of OA-NO2-induced HIF-1a upregulation using the ERK1/2 inhibitor, UO126 (10 mM), and the MAPK/ ERK kinase (MEK)-specific inhibitor PD98059 (50 mM). Pre- treatment of BAECs with UO126 or PD98059 decreased HIF-1a levels by approximately 50% relative to cells treated with OA-NO2 PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31056