addition, AMPK decreases fatty acid incorporation into glycerolipids in some tissues, either secondary to its effect on fatty acid oxidation or via phosphorylation and inhibition of sn- glycerophosphate acyltransferase, the first committed enzyme in diacylglycerol and triglyceride synthesis [56]. Furthermore, endothelial AMPK activity may inhibit glycerol-3-phosphate acyltransferase, which is required for de novo synthesis of diacylglycerol [56]. Thus, AMPK may lessen endothelial diacylglycerol production (and thus protein kinase C activation) both by reducing the availability of the FFA substrates required for this synthesis and by directly inhibiting the enzyme that catalyzes it. In conclusion, we have demonstrated for the first time that NO2-UFAs activate AMPK in endothelial cells by a mechanism that depends on an increase in HO-1 followed by HIF-1a protein expression and Ca2+/CaMKKb activation. Our results also indicate that AMPK activity is required for eNOS/NO produc- tion in endothelial cells (Figure 7F). The present study further suggests that AMPK activation by nitrated lipids may play an essential role in compensating or protecting vascular endothelial function against vascular injury in obesity-related dyslipidemia. AMPK might be a valid therapeutic target for treating vascular disorders in obesity and type II diabetes. Materials and Methods Materials BAECs and cell culture media were obtained from Clonetics Inc. (Walkersville, MD). HUVECs and cell culture media were purchased from Cascade Biologics (Portland, OR). FFA-free bovine serum albumin (BSA) and oleic acid were obtained from Sigma (St. Louis, MO). ZnBG was purchased from Porphyrin Products, Inc. (Logan, UT). OA-NO2 was obtained from Cayman Chemical (Ann Arbor, MI), and AICAR was purchased from Toronto Research Chemicals (Toronto, Canada). The MEK inhibitors PD 98059 and UO 126 were obtained from Calbiochem (La Jolla, CA). 3-(59-hydroxymethyl-29-furyl)-1-benzylindazole (YC-1) was purchased from AG Scientific Inc. (San Diego, CA). Antibodies against phospho-ACC (Ser79), phospho-AMPK (Thr172), AMPK, phospho-LKB1 (Ser428), LKB1, and phos- pho-eNOS (Ser1177) were purchased from Cell Signaling (Beverly, MA). The antibodies against ACC were obtained from Alpha Diagnostic International (San Antonio, TX). All other chemicals and organic solvents were of the highest grade and were obtained from Sigma. Cell culture and adenoviral infection BAECs were grown in EBM supplemented with 2% fetal bovine serum and growth factors. HUVECs were maintained in Medium 200 supplemented with a low-serum growth supplement before use. All culture media were supplemented with both penicillin (100 U/ml) and streptomycin (100 mg/ml). Cells between passages 5 and 10 were used for all experiments. All cells were incubated in a humidified atmosphere of 5% CO2/ 95% air at 37uC. BAECs were infected with adenovirus encoding a dominant-negative mutant form of AMPKa (Ad-DN-AMPK) or green fluorescence protein (Ad-GFP) as a control. Infections were performed in 80% confluent cultures of BAECs in media containing 0.1% fetal bovine serum and recombinant adenovirus at the indicated multiplicity of infection (MOI). Cultures were incubated with adenoviruses for 48 h before experimentation. Using these conditions, infection efficiency was typically at least 80%, as determined by GFP expression. AMPK-Dependent Angiogenesis by Nitrated Lipids SiRNA-mediated gene silencing in endothelial cells HUVECs or BAECs were transfected with LKB1 siRNA, HO-1 siRNA, HIF–1a siRNA, CaMKKb, or the corresponding scrambled siRNA (negative control) for 48 h using Lipofectami- neTM 2000 (Invitrogen) according to the manufacturer’s instruc- tions. Infected cells were starved in serum-free medium for 6 h, then exposed to the indicated concentrations of OA-NO2 or vehicle for 24 h. Measurement of NO production For NO detection, BAECs grown in 24-well plates were incubated for 30 min in the presence of 15 mM 4,5-diaminofluor- escein diacetate (DAF-2 DA) in PBS or in PBS alone (control) in the dark at 37uC. Cells were then washed with PBS to remove excess DAF-2 DA, and the change in fluorescence over 15 min was measured with excitation and emission wavelengths of 485 and 530 nM, respectively, at room temperature using a microplate reader (FL 600, Bio-Tek). Changes in fluorescence were also visualized with a fluorescence microscope (Olympus IX71), and images were captured for analysis [57]. Measurement of intracellular Ca2+ [Ca2+]i was measured using a Fluo-4 NW kit from Invitrogen according to the manufacturer’s instructions. In brief, BAECs were treated with OA-NO2, control or HIF-1a siRNA, YC-1, or BAPTA-AM. The culture medium was then aspirated, cells were washed once with Hepes buffer (pH 7.4), and 1 ml of Hepes buffer with fluorescent dye was added to the cells. After the cells were incubated for 30 min, the fluorescence intensity was measured with excitation and emission wavelengths of 485 and 520 nM, respectively. Western blot analysis BAECs, HUVECs, or Hela-S3 cells were lysed in cold RIPA buffer. Protein concentrations were determined using a bicinch- oninic acid protein assay system (Pierce, Rockford, IL). Proteins were analyzed by western blotting with ECL-Plus detection as described previously [58]. Relative PPARc protein expression was measured in HUVEC nuclear extracts as previously detailed [59]. AMPK activity assay AMPK activity was assayed in the presence and absence of AMP (200 mM) using the SAMS peptide, as previously described [60]. AMPK activity was calculated by determining the difference in activity between both conditions. Statistical analysis Statistical comparisons of vasodilation were performed using a two-way analysis of variance (ANOVA), and intergroup differ- ences were analyzed using Bonferroni’s post-hoc test. Time-course studies were analyzed using a repeated-measure ANOVA. All other results were analyzed using a one-way ANOVA. Values are expressed as mean 6 SD. P-values less than 0.05 were considered significant. Author Contributions Conceived and designed the experiments: YW MZ. Performed the experiments: YW YD PS. Analyzed the data: YW YD PS MZ. Wrote the paper: YW MZ. PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31056