Aspirin prevents resistin-induced endothelial dysfunction by modulating AMPK, ROS, and Akt/eNOS signaling Hsiu-Chung Ou, PhD, Wen-Jane Lee, PhD, Ching-Mei.

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Aspirin prevents resistin-induced endothelial dysfunction by modulating AMPK, ROS, and Akt/eNOS signaling Hsiu-Chung Ou, PhD, Wen-Jane Lee, PhD, Ching-Mei Wu, MS, Judy Fuh-Meei Chen, PhD, Wayne Huey-Herng Sheu, MD, PhD Journal of Vascular Surgery Volume 55, Issue 4, Pages 1104-1115 (April 2012) DOI: 10.1016/j.jvs.2011.10.011 Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 1 Effects of aspirin on resistin-induced reactive oxygen species (ROS) generation in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were pretreated for 2 hours with various concentrations of aspirin (10-500 μg/mL), followed by treatment with 100 ng/mL resistin for 48 hours. At the end of treatment, HUVECs were incubated with the superoxide-sensitive fluorescent probe dihydroethidium (DHE; 10 μM) for 1 hour. A, Fluorescence images show the ROS level in control cells (left) and in HUVECs stimulated with resistin (middle) in the presence of 500 μg/mL aspirin (right). B, Fluorescence intensity of HUVECs was measured with a fluorescence microplate reader. Fluorescence distribution of DHE oxidation was expressed as a percentage of increased intensity. The activities of superoxide dismutase (SOD) (C) and catalase (D) in HUVECs stimulated with resistin in the absence or presence of indicated concentrations of aspirin were determined. Data are expressed as the mean ± SEM of three independent analyses. #P < .05 vs untreated control; *P < .05 compared with resistin treatment. Journal of Vascular Surgery 2012 55, 1104-1115DOI: (10.1016/j.jvs.2011.10.011) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 2 Aspirin attenuated the level of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase membrane assembly. Human umbilical vein endothelial cells (HUVECs) were pretreated for 2 hours with the indicated concentrations of aspirin followed by stimulation with resistin (100 ng/mL) for 1 hour (A-C) or 24 hours (D-F). Preparation of membrane and cytosolic proteins is described in the Materials and Methods section. The levels of cytosolic protein and membrane protein were normalized to the levels of β-actin and flotillin-1, respectively. Representative Western blots and summary data showing that aspirin protected against resistin-induced p47phox and Rac-1 translocation to the plasma membrane, as well as gp91phox and p22phox expression. The values represent means ± SEM from three separate experiments. #P < .05 vs untreated control; *P < .05 compared with resistin treatment. DPI, Diphenyleneiodonium. Journal of Vascular Surgery 2012 55, 1104-1115DOI: (10.1016/j.jvs.2011.10.011) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 3 Aspirin prevented resistin-induced PKC-α translocation via AMP-activated protein kinase (AMPK) phosphorylation. Human umbilical vein endothelial cells (HUVECs) were pretreated with aspirin (10-500 μg/mL) or diphenyleneiodonium (DPI; 5 μM) or 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) (500 μM) for 2 hours followed by exposure to resistin (100 ng/mL) for a further 1-hour period. Representative Western blots and summary data show that aspirin protected against resistin-induced AMPK dephosphorylation (A and B) and PKC-α translocation to the plasma membrane (C and D). In AMPK knockdown experiments, HUVECs were transfected with AMPKα1 siRNA for 24, 48, and 72 hours. Protein expression of AMPK (E) and phosphorylated acetyl-CoA carboxylase (p-ACC) (F) were checked by Western blots. The effects of aspirin on resistin-induced membrane translocation of PKC-α and p47phox were abolished in cells that had been transfected with AMPK siRNA for 48 hours (G-I). Preparation of membrane and cytosolic proteins is described in the Materials and Methods section. The levels of cytosolic and membrane protein were normalized to the levels of β-actin and flotillin-1, respectively. The values represent means ± SEM of three separate experiments. #P < .05 vs untreated control; *P < .05 compared with resistin treatment; &P < .05 vs resistin + aspirin treatment. Journal of Vascular Surgery 2012 55, 1104-1115DOI: (10.1016/j.jvs.2011.10.011) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 4 The protective effects of aspirin involve AMP-activated protein kinase (AMPK)/Akt/endothelial nitric oxide synthase (eNOS) activation. Human umbilical vein endothelial cells (HUVECs) were pretreated for 2 hours with the indicated concentrations of aspirin, aspirin plus Compound C, or 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) followed by resistin (100 ng/mL) for an additional 1-hour period. In a parallel experiment, HUVECs were transfected with AMPK siRNA for 48 hours before treatment of resistin. Western blot analysis for Akt, eNOS, phospho-Akt (p-Akt), and phospho-eNOS (p-eNOS) (A). The values represent means ± SEM of three separate experiments (B and C). #P < .05 vs untreated control; *P < .05 compared with treatment; &P < .05 vs resistin + aspirin treatment. Journal of Vascular Surgery 2012 55, 1104-1115DOI: (10.1016/j.jvs.2011.10.011) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 5 Effect of aspirin on p38 mitogen-activated protein kinase (p38 MAPK)-mediated NF-κB activation caused by resistin. Pretreatment of human umbilical vein endothelial cells (HUVECs) with indicated concentrations of aspirin, or an inhibitor of p38 MAPK (SB203580), attenuated p38 MAPK activation, IκB-α degradation, and NF-κB activation caused by resistin. HUVECs were pretreated with various concentrations of aspirin or SB203580 for 2 hours, followed by incubation with resistin (100 ng/mL) for 1 hour. At the end of the incubation period, cells were lysed and proteins were analyzed by Western blot. Protein levels of phosphor-p38 MAPK, NF-κB, and IκB-α were normalized to the level of total p38 MAPK, PCNA, and β-actin, respectively (A). Data illustrated on the graph bars represent the mean ± SEM of three separate experiments (B-D). #P < .05 vs untreated control; *P < .05 compared to resistin treatment. Journal of Vascular Surgery 2012 55, 1104-1115DOI: (10.1016/j.jvs.2011.10.011) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 6 Aspirin suppressed the resistin-induced downregulation of endothelial nitric oxide synthase (eNOS), upregulation of inducible NO synthase (iNOS), the nitration of tyrosine residues (A and B), and resistin-enhanced nitrous oxide (NO) release (C). Human umbilical vein endothelial cells (HUVECs) were pretreated for 2 hours with the indicated concentrations of aspirin followed by stimulation with resistin (100 ng/mL) for 48 hours. At the end of the incubation period, levels of eNOS, iNOS, and nitrotyrosine protein (normalized to the levels of β-actin) were determined by immunoblotting; content of NO was assayed using Griess reagent. The values represent means ± SEM from three separate experiments. #P < .05 vs untreated control; *P < .05 compared to resistin treatment. Journal of Vascular Surgery 2012 55, 1104-1115DOI: (10.1016/j.jvs.2011.10.011) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 7 Effect of aspirin on resistin-induced mRNA and protein expression of inflammatory cytokines, adhesion molecules, and monocytic cells adherence. Human umbilical vein endothelial cells (HUVECs) were pretreated for 2 hours with or without indicated concentrations of aspirin (10, 100, 300, and 500 μg/mL) followed by resistin (100 ng/mL) incubation for 48 hours. mRNA levels of interleukin (IL)-6, IL-8 (A), and intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) as well as E-selectin (B) were determined by real-time polymerase chain reaction (PCR). Protein levels of IL-6, IL-8 (C), and ICAM-1, VCAM-1, as well as E-selectin (D) were determined by enzyme-linked immunosorbent assay (ELISA). E and F, Cells were pretreated with each inhibitor 1 hour before aspirin treatment and then incubated for 48 hours with resistin (100 ng/mL). Resistin-induced adhesiveness of monocytic leukemia cells (THP-1) to HUVECs was measured as described in the Materials and Methods section. The values represent means ± SEM from three separate experiments. #P < .05 vs untreated control; *P < .05 compared with resistin treatment; &P < .05 vs resistin + aspirin treatment. Journal of Vascular Surgery 2012 55, 1104-1115DOI: (10.1016/j.jvs.2011.10.011) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 8 Summary and working hypothesis showing the signaling cascades involved in the attenuation of inflammatory responses in resistin-exposed cells treated with aspirin. The → indicates activation or induction, and ⊣ indicates inhibition or blockade. Resistin has been reported to be involved in the inflammatory process, which is a common feature in metabolic syndrome, insulin resistance status, and vascular diseases. This study underscores the potential clinical benefits and application of aspirin in prevention of obese-associated vascular dysfunction. AMPK, AMP-activated protein kinase; eNOS, endothelial nitric oxide synthase; ICAM, intercellular adhesion molecule; IL, interleukin; iNOS, inducible nitric oxide snythase; NADPH, nicotinamide adenine dinucleotide phosphate; p38 MAPK, p38 mitogen-activated protein kinase; ROS, reactive oxygen species; SOD, superoxide dismutase; VCAM, vascular cell adhesion molecule. Journal of Vascular Surgery 2012 55, 1104-1115DOI: (10.1016/j.jvs.2011.10.011) Copyright © 2012 Society for Vascular Surgery Terms and Conditions