title: Aspirin prevents resistin-induced endothelial dysfunction by modulating AMPK, ROS, and Akt/eNOS signaling.
abstract: BACKGROUND
  Resistin, an adipocytokine, plays a potential role in cardiovascular disease and may contribute to increased atherosclerotic risk by modulating the activity of endothelial cells. A growing body of evidence suggests that aspirin is a potent antioxidant. We investigated whether aspirin mitigates resistin-induced endothelial dysfunction via modulation of reactive oxygen species (ROS) generation and explored the role that AMP-activated protein kinase (AMPK), a negative regulator of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, plays in the suppressive effects of aspirin on resistin-induced endothelial dysfunction.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) were pretreated with various doses of aspirin (10-500 μg/mL) for 2 hours and then incubated with resistin (100 ng/mL) for an additional 48 hours. Fluorescence produced by the oxidation of dihydroethidium (DHE) was used to quantify the production of superoxide in situ; superoxide dismutase (SOD) and catalase activities were determined by an enzymatic assay; and protein levels of AMPK-mediated downstream signaling were investigated by Western blot.
  
  
  RESULTS
  Treatment of HUVECs with resistin for 48 hours resulted in a 2.9-fold increase in superoxide production; however, pretreatment with aspirin resulted in a dose-dependent decrease in production of superoxide (10-500 μg/mL; n = 3 experiments; all P < .05). Resistin also suppressed the activity of superoxide dismutase and catalase by nearly 50%; that result, however, was not observed in HUVECs that had been pretreated with aspirin at a concentration of 500 μg/mL. The membrane translocation assay showed that the levels of NADPH oxidase subunits p47(phox)and Rac-1 in membrane fractions of HUVECs were threefold to fourfold higher in cells that had been treated with resistin for 1 hour than in untreated cells; however, pretreatment with aspirin markedly inhibited resistin-induced membrane assembly of NADPH oxidase via modulating AMPK-suppressed PKC-α activation. Application of AMPKα1-specific siRNA resulted in increased activation of PKC-α and p47(phox). In addition, resistin significantly decreased AMPK-mediated downstream Akt/endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling and induced the phosphorylation of p38 mitogen-activated protein kinases, which in turn activated NF-κB-mediated inflammatory responses such as the release of interleukin (IL)-6 and IL-8, the overexpression of adhesion molecules, and stimulation of monocytic THP-1 cell attachment to HUVECs (2.5-fold vs control; n = 3 experiments). Furthermore, resistin downregulated eNOS and upregulated inducible NO synthase (iNOS) expression, thereby augmenting the formation of NO and protein nitrosylation. Pretreatment with aspirin, however, exerted significant cytoprotective effects in a dose-dependent manner (P < .05).
  
  
  CONCLUSIONS
  Our findings suggest a direct connection between adipocytokines and endothelial dysfunction and provide further insight into the protective effects of aspirin in obese individuals with endothelial dysfunction.
authors.affiliation: Department of Physical Therapy and Graduate, Institute of Rehabilitation Science, China Medical University, Taichung, Taiwan, Republic of China.
authors.forename: Hsiu-Chung
authors.initials: HC
authors.lastname: Ou
authors.forename: Wen-Jane
authors.initials: WJ
authors.lastname: Lee
authors.forename: Ching-Mei
authors.initials: CM
authors.lastname: Wu
authors.forename: Judy Fuh-Meei
authors.initials: JF
authors.lastname: Chen
authors.forename: Wayne Huey-Herng
authors.initials: WH
authors.lastname: Sheu
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D000704:Analysis of Variance; D001241:Aspirin / Q000494:pharmacology*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D004334:Drug Administration Schedule; D004797:Enzyme-Linked Immunosorbent Assay; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D009249:NADP / Q000187:drug effects / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering / Q000032:analysis; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012016:Reference Values; D052243:Resistin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D017077:Culture Media, Conditioned; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D052243:Resistin; D009249:NADP; D019001:Nitric Oxide Synthase; D051057:Proto-Oncogene Proteins c-akt; D055372:AMP-Activated Protein Kinases; D001241:Aspirin